Evaluation of micro-energy dispersive X-ray fluorescence and histochemical tests for aluminium detection in plants from High Altitude Rocky Complexes, Southeast Brazil

NAIARA V. CAMPOS TIAGO A.R. PEREIRA MARIANA F. MACHADO MARCELO B.B. GUERRA GLÁUCIA S. TOLENTINO JOSIANE S. ARAÚJO MAÍRA Q. REZENDE MARIA CAROLINA N.A. DA SILVA CARLOS E.G.R. SCHAEFER About the authors

Abstracts

The soils developed under High Altitude Rocky Complexes in Brazil are generally of very low chemical fertility, with low base saturation and high exchangeable aluminium concentration. This stressful condition imposes evolutionary pressures that lead to ecological success of plant species that are able to tolerate or accumulate high amounts of aluminium. Several analytical methods are currently available for elemental mapping of biological structures, such as micro-X-ray fluorescence (μ-EDX) and histochemical tests. The aim of this study was to combine μ-EDX analysis and histochemical tests to quantify aluminium in plants from High Altitude Rocky Complexes, identifying the main sites for Al-accumulation. Among the studied species, five showed total Al concentration higher than 1000 mg kg−1. The main Al-hyperaccumulator plants,Lavoisiera pectinata, Lycopodium clavatumand Trembleya parviflora presented positive reactions in the histochemical tests using Chrome Azurol and Aluminon. Strong positive correlations were observed between the total Al concentrations and data obtained by μ-EDX analysis. The μ-EDX analysis is a potential tool to map and quantify Al in hyperaccumulator species, and a valuable technique due to its non-destructive capacity. Histochemical tests can be helpful to indicate the accumulation pattern of samples before they are submitted for further μ-EDX scrutiny.

Al-hyperaccumulator plants; aluminon; chrome azurol; High Altitude Rocky Complexes; μ-EDX


Solos associados aos Complexos Rupestres de Altitude no Brasil destacam-se pela baixa fertilidade química, baixa saturação de bases e elevados teores de alumínio trocável. Esta condição estressante impõe pressões evolutivas que determinam o sucesso ecológico de espécies capazes de tolerar ou acumular grandes quantidades de alumínio. Vários métodos analíticos são utilizados para mapeamento de elementos químicos em estruturas biológicas, como microfluorescência de raios-X (μ-EDX) e testes histoquímicos. O objetivo do presente trabalho foi combinar a μ-EDX e testes histoquímicos para quantificar o teor de Al em espécies vegetais de Complexos Rupestres de Altitude, e identificar os principais sítios de bioacumulação de Al. Cinco das espécies investigadas apresentaram concentração total de Al maior que 1000 mg kg−1. As principais hiperacumuladoras de Al,Lavoisiera pectinata, Lycopodium clavatume Trembleya parviflora, apresentaram reação positiva nos testes histoquímicos com Chrome Azurol e Aluminon. Alta correlação positiva foi observada entre as concentrações totais de Al e as magnitudes de sinal obtidas por μ-EDX. A análise com o uso da μ-EDX mostrou-se uma ferramenta promissora para mapear e quantificar Al em espécies hiperacumuladoras, constituindo uma importante técnica não destrutiva. Testes histoquímicos podem ser úteis na identificação de padrões de acumulação de Al em amostras vegetais antes de serem submetidas a uma minuciosa análise com a μ-EDX.

plantas hiperacumuladoras de Al; aluminon; chrome azurol; Complexos Rupestres de Altitude; μ-EDX


INTRODUCTION

The soils of tropical and subtropical regions commonly exhibit acidic properties due to intense leaching, which results in removal of negative charges and retention of compounds containing iron and aluminium (Echart and Molina 2001Echart C and Molina SC. 2001. Fitotoxicidade do alumínio: efeitos, mecanismo de tolerância e seu controle genético. Ciênc Rural 31: 531-541.). About 30 % of the world's soils are acidic, with pH ≤ 5.5, presenting low levels of organic matter and base saturation, and high levels of exchangeable aluminium (Al3+) (Hartwig et al. 2007Hartwig I, Oliveira AC, Carvalho FIF, Bertan I, Silva JAG, Schmidt DAM, Valério IP, Maia LC, Fonseca DNR and Reis CES. 2007. Mecanismos associados à tolerância ao alumínio em plantas. Semina Ciências Agrárias 28: 219-228.). In Brazil, soils derived from granitic and gneiss rocky outcrops of the Serra da Mantiqueira are associated with high Al3+ contents (Benites et al. 2007Benites VM, Schaefer CEGR, Simas FNB and Santos HG. 2007. Soils associated with rock outcrops in the Brazilian mountain ranges Mantiqueira and Espinhaço. Rev Bras Bot 30: 569-577.).

High soil acidity increases aluminium availability, which, in turn, can affect Al-sensitive plants causing both root growth inhibition and thickening of root epidermis (Ciamporová 2002Ciamporová M. 2002. Morphological and Structural Responses of Plant Roots to Aluminium at Organ, Tissue, and Cellular Levels. Biol Plant 45: 161-171.,Vitorello et al. 2005Vitorello VA, Capaldi FR and Stefanuto VA. 2005. Recent advances in aluminum toxicity and resistance in higher plants. Braz J Plant Physiol 17: 129-143.). Low nutrient availability combined with high aluminium concentrations in soil solution act as an important environmental stress agent (Larcher 2000Larcher W. 2000. Ecofisiologia vegetal. São Carlos: Rima, 531 p., Grime 2001Grime JP. 2001. Plant strategies, vegetation processes, and ecosystem properties, 2nd ed., Chichester: John Wiley & Sons Ltd, 456 p.). The tolerance to high Al3+ concentrations is an ecological attribute that permits the occupation of a site qualified as inappropriate for Al-sensitive species (Jansen et al. 2002aJansen S, Broadley MR, Robbrecht E and Smets E. 2002a. Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. Bot Rev 68: 235-269.). Some plants living in these environments can accumulate more than 1000 mg kg−1 of Al in their tissues, being called hyperaccumulator species (Baker 1981Baker AJM. 1981. Accumulators and excluders: Strategies in the response of plants to heavy metals. Journal of Plant Nutrition 3: 643-654.). Hyperaccumulator plants have mechanisms of aluminium resistance, such as synthesis of chelator agents and turnover of roots and leaves which have already reached high levels of aluminium (Cuenca and Herrera 1987Cuenca G and Herrera R. 1987. Ecophysiology of aluminium in terrestrial plants, growing in acid and aluminium-rich tropical soils. Ann Soc R Zool Belg 117: 57-74., Cuenca and Medina 1990Cuenca G and Medina E. 1990. Aluminium tolerance in trees of a tropical cloud forest. PI & Soil 125: 169-175.).

Melastomataceae, Rubiaceae, Asteraceae, Vochysiaceae and Myrtaceae contain a great number of taxa whose life histories are related to soils presenting high Al contents (Chenery and Sporne 1976Chenery EM and Sporne KR. 1976. A note on the evolutionary status of aluminium-accumulators among Dicotyledons. New Phytologist 76: 551-554., Haridasan 1988Haridasan M. 1988. Performance of Miconia albicans (SW.) Triana, an aluminum-accumulating species, in acidic and calcareous soils. Comm Soil Sci Plant Anal 19: 1091-1103., Jansen et al. 2000Jansen S, Dessein S, Piesschaert R, Robbrecht E and Smets E. 2000. Aluminium accumulation in leaves of Rubiaceae: Systematic and phylogenetic implications. Ann Bot 85: 91-101., 2002aJansen S, Broadley MR, Robbrecht E and Smets E. 2002a. Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. Bot Rev 68: 235-269., bJansen S, Watanabe T and Smets E. 2002b. Aluminium accumulation in leaves of 127 species in Melastomataceae, with comments on the order Myrtales. Ann Bot 90: 53-64.). Lycopodiaceae and Pteridaceae were previously described as pertaining to the aluminium accumulator families (Church 1888Church AH. 1888. On the occurrence of aluminium in certain vascular cryptogams. P R Soc London 44: 121-129., Olivares et al. 2009Olivares E, Pena E, Marcano E, Mostacero J, Aguiar G, Benitez M and Rengifo E. 2009. Aluminium accumulation and its relationship whit mineral plant nutrients in 12 pteridophytes from Venezuela. Environ Exp Bot 65: 132-141.). The chelation mechanism of these species is based on Al3+ retention by organic acids in celular compartments such as cell wall and vacuole (Taylor 1991Taylor GJ. 1991. Current views of the aluminum stress response: the physiological basis of tolerance. In: RANDALL DD, BLEVINS DG and MILES CD (Eds), Current Topics in Plant Biochemistry Physiology, Columbia: Missouri, v.10, p. 57-93., Delhaize and Ryan 1995Delhaize E and Ryan PR. 1995. Aluminum toxicity and tolerance in plants. Plant Physiol 107: 315-321.,Shen et al. 2002Shen R, Ma J, Kyo M and Iwashita T. 2002. Compartmentation of aluminium in leaves of an Al-accumulator, Fagopyrum esculentum Moench. Planta 215: 394-398.).

The study of metal distribution patterns in plant tissues can clarify accumulation processes in tolerant species, simultaneously quantifying and mapping the content of chemical elements (Moradi et al. 2010Moradi AB, Swoboda S, Robinson B, Prohaska T, Kaestner A, Oswald SE, Wenzel WW and Schulin R. 2010. Mapping of nickel in root cross sections of the hyperaccumulator plant Berkheya coddii using laser ablation ICPMS. Environ Exp Bot 69: 24-31.). Methods commonly used for mapping studies in biological tissues are based on μ-EDX or Synchrotron radiation X-ray fluorescence (West et al. 2009West M, Ellis AT, Potts PJ, Streli C, Vanhoof C, Wegrzynek D and Wobrauschek P. 2009. Atomic spectrometry update. X-Ray fluorescence spectrometry. J Anal Spectrom 24: 1289-1326., Majumdar et al. 2012Majumdar S, Peralta-Videa JR, Castillo-Michel H, Hong J, Rico CM and Gardea-Torresdey JL. 2012. Applications of synchrotron μ-XRF to study the distribution of biologically important elements in different environmental matrices: A review. Anal Chim Acta 755: 1-16.), micro-proton-induced X-ray emission (Lyubenova et al. 2012Lyubenova L, Pongrac P, Vogel-Mikuš K, Mezek GK, Vavpetič P, Grlj N, Kump P, Nečemer M, Regvar M, Pelicon P and Schröder P. 2012. Localization and quantification of Pb and nutrients in Typha latifolia by micro-PIXE. Metallomics 4: 333-341.), and also laser-based methods such as laser ablation inductively coupled plasma mass spectrometry (Guerra et al. 2011Guerra MBB, Amarasiriwardena D, Schaefer CEGR, Pereira CD, Spielmann AA, Nóbrega JA and Pereira-Filho ER. 2011. Biomonitoring of lead in Antarctic lichens using laser ablation inductively coupled plasma mass spectrometry. J Anal At Spectrom 26: 2238-2246., Qin et al. 2011Qin Z, Caruso JA, Lai B, Matush A and Becker JS. 2011. Trace metal imaging with high spatial resolution: Applications in biomedicine. Metallomics 3: 28-37.) and laser induced breakdown spectroscopy (Galiová et al. 2007Galiová M, Kaiser J, Novotný K, Samek O, Reale L, Malina R, Páleníková K, Liška M, Čudek V, Kanický V, Otruba V, Poma A and Tucci A. 2007. Utilization of laser induced breakdown spectroscopy for investigation of the metal accumulation in vegetal tissues. Spectrochim Acta B 62: 1597-1605., Santos Jr. et al. 2012, Piñon et al. 2013Piñon V, Mateo MP and Nicolas G. 2013. Laser-induced breakdown spectroscopy for chemical mapping of materials. Appl Spectrosc Rev 48: 357-383.).

X-ray fluorescence analysis (XRF) is a fast and non-destructive method which has several applications (Saisho and Hashimoto 1996Saisho H and Hashimoto H. 1996. X-ray fluorescense analysis. In: SAISHO H AND GOHSHI Y (Eds), Applications of Synchontron Radiation to Materials Analysis. Amsterdam: Elsevier, p. 79-169., West et al. 2009West M, Ellis AT, Potts PJ, Streli C, Vanhoof C, Wegrzynek D and Wobrauschek P. 2009. Atomic spectrometry update. X-Ray fluorescence spectrometry. J Anal Spectrom 24: 1289-1326.). Although the common detection limit of the XRF technique ranges from mg kg−1 to % ww−1 (Saisho and Hashimoto 1996Saisho H and Hashimoto H. 1996. X-ray fluorescense analysis. In: SAISHO H AND GOHSHI Y (Eds), Applications of Synchontron Radiation to Materials Analysis. Amsterdam: Elsevier, p. 79-169.), it can be successfully applied to hyperaccumulator species tissues (Memon et al. 1981Memon AR, Chino M and Yatazawa M. 1981. Microdistribution of aluminum and manganese in the tea leaf tissues as revealed by X-ray microanalyzer. Commun Soil Sci Plant Anal 12: 441-452., Cuenca et al. 1991Cuenca G, Herrera R and Mérida T. 1991. Distribution of aluminium in accumulator plants by X-ray microanalysis in Richeria grandis Vahl leaves from a cloud forest in Venezuela. Plant Cell Environ 14: 437-441., Robinson et al. 2003Robinson BH, Lombi E, Zhao FJ and McGrath SP. 2003. Uptake and distribution of nickel and other metals in the hyperaccumulator Berkheya coddii. New Phytologist 158: 279-285.,Broadhurst et al. 2004Broadhurst CL, Chaney RL, Angle JS, Erbe EF and Maugel TK. 2004. Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale. Plant and Soil 265: 225-242., Berazain et al. 2007Berazain R, De La Fuente V, Rufo L, Rodriguez N, Amils R, Diez-Garretas B, Sanchez-Mata D and Asensi A. 2007. Nickel localization in tissues of different hyperaccumulator species of Euphorbiaceae from ultramafic areas of Cuba. Plant and Soil 293: 99-106., Turnau et al. 2007Turnau K, Henriques FS, Anielska T, Renker C and Buscot F. 2007. Metal uptake and detoxification mechanisms in Erica andevalensis growing in a pyrite mine tailing. Environ Exp Bot 61: 117-123., Tolrà et al. 2011). In order to perform quantitative measurements, calibration can be done by the linear relationship between the intensity of X-ray emission of a target element and its concentration previously determined by a reference method. The quality of the XRF data can be evaluated by the correlation between the XRF intensity and the reference values using figures of merit such as linear correlation factor, standard error of prediction (SEP), confidence intervals and bias (Paltridge et al. 2012Paltridge NG, Milham PJ, Ortiz-Monasterio JI, Velu G, Yasmin Z, Palmer LJ, Guild GE and Stangoulis JCR. 2012. Energy-dispersive X-ray fluorescence spectrometry as a tool for zinc, iron and selenium analysis in whole grain wheat. Plant Soil 361: 261-269. [10.1007/s11104-012-1423-0]).

Applying histochemical and chemical techniques for detection of compounds of interest, such as potentially toxic metals, can complement the data obtained from X-ray microanalysis, since they are useful for locating discrete quantities of chemical elements in biological tissues (Pearse 1972Pearse AGE. 1972. Histochemistry: theoretical and applied, Vol. II, 3rd ed., Churchill Livingstone, Edinburgh., 1988Pearse AGE. 1988. Histochemistry: theoretical and applied, Vol. I, 4th ed., Longman, London., Krishnamurthy 1998Krishnamurthy KV. 1998. Methods in plant histochemistry. S.Viswanathan, Chennai.). Several reagents are used in Al histolocalization, such as Hematoxylin, Aluminon, Chrome Azurol, Pyrocatecol and Azurine (Baker 1962Baker JR. 1962. Experiments on the action of mordants 2. Aluminum-haematein. Q J Micr Sci 103: 493-517., Denton et al. 1984Denton J, Freemont AJ and Ball J. 1984. Detection and distribution of aluminium in bone. J Clin Pathol 37: 136-142., Clark and Krueger 1985Clark RA and Krueger GL. 1985. Review Article Aluminon: Its Limited Application as a Reagent for the Detection of Aluminum Species. J Histochem Cytochem 33: 729-732., Haridasan et al. 1986Haridasan M, Paviani TI and Schiavini I. 1986. Localization of aluminium in the leaves of some aluminium-accumulating species. Plant Soil 94: 435-437., Cotta et al. 2008Cotta MG, Andrade LRM De, Geest AJV, Gomes ACMM, Souza CMD De, Almeida JD and Barros LMG. 2008. Diferenças entre hematoxilina e aluminon na detecção de alumínio em tecidos foliares de plantas nativas do Cerrado. IX Simpósio Nacional Cerrado. Desafios e estratégias para o equilíbrio entre sociedade, agronegócio e recursos naturais. 12 a 17 de outubro de 2008, Brasília (DF).).

In this context, this study aimed to combine μ-EDX analysis and histochemical tests to quantify aluminium in plants from High Altitude Rocky Complexes, characterizing, furthermore, the main sites for Al-accumulation in shoots.

According to our knowledge this is the first study reporting aluminium detection in hyperaccumulator plants from High Altitude Rocky Complexes using both μ-EDX apparatus and histochemical tests.

MATERIALS AND METHODS

Study Area

Serra do Brigadeiro State Park

The Serra do Brigadeiro State Park is located in Araponga (State of Minas Gerais, Brazil, 20°40′ S and 42° 26′ W) in Serra da Mantiqueira massif. According to Köeppen, the climate is mesothermal (CWb). The mean annual rainfall and air temperature are about 1500 mm and 15 °C, respectively (Benites et al. 2001Benites VM, Schaefer CEGR, Mendonça ES and Martin-Neto L. 2001. Caracterização da matéria orgânica e micromorfologia de solos sob Campos de Altitude no Parque Estadual da Serra do Brigadeiro. Rev Bras Ciênc Solo 25: 661-674.).

The High Altitude Rocky Complexes are located in the highest points of the Serra da Mantiqueira massif, in areas above 1500 m, being associated with igneous and metamorphic parent materials (Vasconcelos 2011Vasconcelos MF. 2011. O que são campos rupestres e campos de altitude nos topos de montanha do Leste do Brasil? Rev Bras Bot 34: 241-246.). The vegetation cover is related to soils with high aluminium saturation and low calcium and magnesium contents (Benites et al. 2001Benites VM, Schaefer CEGR, Mendonça ES and Martin-Neto L. 2001. Caracterização da matéria orgânica e micromorfologia de solos sob Campos de Altitude no Parque Estadual da Serra do Brigadeiro. Rev Bras Ciênc Solo 25: 661-674.).

Studied Species

Ten plant species were sampled: Marcetia taxifolia,Lavoisiera pectinata, Tibouchina heteromalla and Trembleya parviflora(Melastomataceae); Baccharis trimera andEremanthus erythropappus (Asteraceae);Nanuza plicata and Vellozia variegata(Velloziaceae); Myrsine umbellata(Myrsinaceae) and Lycopodium clavatum(Lycopodiaceae). Samples of three individuals of each species were collected. Soil and vegetation classification of sampling sites are described inTable I (B.V. Tinti et al., unpublished data).

TABLE I
Soils and vegetation types studied at the Serra do Brigadeiro State Park.

AL-Localization in Plant Tissues

Samples of stems and leaves from all plant species were fixed using a solution composed of formaldehyde and acetic acid (FAA) 50 % (v v−1), dehydrated through an ethanol series (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York: Mc Graw Hill, 532 p.) and freehand sectioned with a razor blade. Subsequently, the sections were submitted to a histochemical test using Chrome Azurol (reaction time: 15 minutes). Sections that showed positive reactions, evidenced by the blue color, were subjected to new tests with Chrome Azurol and Aluminon for 30 minutes. For Aluminon test the intense red color is identified as a positive reaction.

Photographs were taken using a light microscope (Olympus AX70TRF, Olympus Optical, Tokyo, Japan) coupled with a U-Photo Camera system (Spot Insightcolour 3.2.0, Diagnostic Instruments inc. New York, USA).

AL-Determination in Plant Material

Determination of total Al concentration using ICP OES

Samples of plant material (n = 10) were oven-dried at 70 °C, until constant weight, and were powdered with the help of a knife mill.

Powdered samples were digested, in triplicate, with nitro-perchloric solution in an electric plate following procedure described by Tedesco et al. (1995)Tedesco MJ, Gianello C, Bissani CA, Bohnen H and Volkweiss SJ. 1995. Análise de solo, plantas e outros materiais. Porto Alegre: UFRGS, 174 p.. A comparative wet-based decomposition procedure based on nitric acid and hydrogen peroxide using a microwave-assisted digestion was also performed as described by Guerra et al. (2013)Guerra MBB, Schaefer CEGR, Carvalho GGA, Souza PF, Santos Jr D, Nunes LC and Krug FJ. 2013. Evaluation of micro-Energy Dispersive X-ray Fluorescence Spectrometry for the Analysis of Plant Materials. J Anal At Spectrom 28: 1096-1101..

Determination of total Al concentration in acid extracts was performed using inductively coupled plasma optical emission spectrometry with dual-view configuration (Perkin Elmer, Shelton, CT, EUA). Operational conditions of ICP OES measurements were described in Table II.

TABLE II
Operational conditions in ICP OES determinations.

μ-EDX analysis

Samples which showed higher Al concentration were selected and analyzed using micro-energy dispersive X-ray fluorescence technique (μ-EDX-1300, Shimadzu, Kyoto, Japan). The usefulness of this technique in determination of total Al concentration and mapping studies on the raw sample material was evaluated.

Pellets of powdered plant samples (particle size lower than 250 μm) were prepared after applying 10 t cm−2(Perkin Elmer, Waltham, MA, EUA) pressure during 5 minutes on 0.15 g of dried material. On each pellet 10 points were randomly selected to be analyzed by μ-EDX apparatus. The operational conditions were described in Table III.

TABLE III
Operational conditions in μ-EDX analysis.

A linear regression model was adjusted to correlate Al total concentration determined by ICP OES and μ-EDX intensity (cps μA−1) as recommended by Guerra et al. 2013Guerra MBB, Schaefer CEGR, Carvalho GGA, Souza PF, Santos Jr D, Nunes LC and Krug FJ. 2013. Evaluation of micro-Energy Dispersive X-ray Fluorescence Spectrometry for the Analysis of Plant Materials. J Anal At Spectrom 28: 1096-1101.. By using this linear regression model, it was possible to evaluate the Al distribution on the aerial parts of pre-selected plants. Samples of leaves, with or without the stem, were oven dried at 70 °C and fixed on paper supports using adhesive tape. For each plant species, different parts of leaves and stem were analyzed and, for each part, five points were randomly selected.

Statistical Analysis

The data was analyzed using analysis of variance (ANOVA), followed by Tukey test at 5% significance level. All analyses were performed using R 2.13 software (R Development Core Team 2006R Development Core Team. 2006. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) and were followed by residual analyses to check for the suitability of the models (Crawley 2007Crawley MJ. 2007. The R Book. Chichester: John Wiley & Sons, 951 p.).

RESULTS

AL-Localization in Plant Tissues

Among the analyzed species, seven presented negative result for Al presence using Chrome Azurol (Myrsine umbellate, Eremanthus erythropappus, Vellozia variegata,Baccharis trimera, Tibouchina heteromalla,Nanuza plicata and Marcetia taxifolia). Otherwise, three species (Lavoisiera pectinata, Trembleya parviflora andLycopodium clavatum) showed a positive reaction for Chrome Azurol and Aluminon, which indicates higher Al concentration in the stem or leaf tissues (Figure 1). The positive reaction to Chrome Azurol was noted in primary walls, epidermal cells, parenchyma and phloem. A negative reaction to Chrome Azurol was observed in secondary cell walls including those of xylem vessels and sclerenchyma fibers. Aluminon results showed the same pattern observed for Chrome Azurol.

Figure 1
Cross sections of leaves of Lavoisiera pectinata (A-C), Trembleya parviflora (D-F) and stem of Lycopodium clavatum (G-I). A, D andG. Tissues treated with Aluminon. B,E and H. Tissues treated with Chrome Azurol. C, F and I. Blank test. Bars length = 100 μm.

The reaction in L. pectinata was markedly evident in the cuticle layer on both sides of the leaf epidermis cells (Figure 1A-C). In T. parviflora a negative reaction to Chrome Azurol was noted in palisade parenchyma cells (Figure 1E) even though a positive reaction to Aluminon had been detected (Figure 1D). A positive reaction to Aluminon was observed around the vascular cylinder in L. clavatum (Figure 1G).

AL-Determination in Plant Material

Total Al concentration

The average of the total Al concentration in plants (mg kg−1) was as follows: Vellozia variegata (216), Myrsine umbellata(297), Nanuza plicata (344),Marcetia taxifolia (641), Baccharis trimera (749), Eremanthus erythropappus (1149), Tibouchina heteromalla (2000), Trembleya parviflora (3878), Lavoisiera pectinata (8589) and Lycopodium clavatum (9049). The following samples: L. clavatum, L. pectinata and T. parviflora were selected for X-ray fluorescence analysis due the highest Al concentrations observed in these species.

μ-EDX results

Micro-energy dispersive X-ray fluorescence analysis revealed significant differences between the studied species after evaluating the data obtained from Al peak intensities (F2.27 = 14.323; p < 0.0001) at 1.49 keV (Kα line). L. clavatum (0.029 cps μA−1) andL. pectinata (0.027 cps μA−1) had the highest intensities and its averages were not significantly different (F2,28 = 0.245, p = 0.62) (Figure 2). T. parviflora had a mean significantly lower (0.0123 cps μA−1). Linear regression model, adjusted in order to verify the correlation between the total Al concentrations and the intensity values, revealed a high linear correlation factor (r2 higher than 0.99).

Figure 2
Micro-energy dispersive X-ray spectrometry data of three different plant species in pellet formats (F2,27= 14.323; p < 0.0001). Different letters represent significant differences of detected intensities.

The highest Al concentrations in leaves, obtained by μ-EDX analysis were observed in L. pectinata(F2,9 = 31.84, p = 0.002). This result, however, underestimate the concentration of Al in L. clavatum when the average concentration in the points were compared with the total Al concentration obtained by ICP OES determination . In L. pectinata and L. clavatum, there were no differences in Al concentrations among the average concentrations of the analyzed regions and the ICP OES results (F2,14 = 0.51, p = 0.61; F2,29 = 0.06, p = 0.81; respectively). In T. parviflora the highest Al concentration was observed in the midvein (F2,32 = 20.00, p < 0.0001) (Figure 3).

Figure 3
Aluminium concentration evaluated by micro-energy dispersive X-ray spectrometry in dry leaves of three different plant species (F2,9 = 31.84; p = 0.002). Different letters represent significant differences of aluminium concentration. A-B: Lavoisiera pectinata. C-D: Trembleya parviflora. E-F: Lycopodium clavatum. A, C andE indicate the selected points analyzed in leaves and stem. Bars length = 1 mm.

DISCUSSION

The occurrence of plant species in soils with high availability of Al3+ suggests physiological mechanisms that define them as stress-tolerant (Chenery and Sporne 1976Chenery EM and Sporne KR. 1976. A note on the evolutionary status of aluminium-accumulators among Dicotyledons. New Phytologist 76: 551-554., Grime 2001Grime JP. 2001. Plant strategies, vegetation processes, and ecosystem properties, 2nd ed., Chichester: John Wiley & Sons Ltd, 456 p., Jansen et al. 2002aJansen S, Broadley MR, Robbrecht E and Smets E. 2002a. Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. Bot Rev 68: 235-269., Ramírez-Rodriguez et al. 2005). Hyperaccumulator plant species can be found in approximately 45 different families, being an intrinsic characteristic to at least 18 of them (Jansen et al. 2002aJansen S, Broadley MR, Robbrecht E and Smets E. 2002a. Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. Bot Rev 68: 235-269.).

Among the sampled species, Lycopodium clavatum,Lavoisiera pectinata, Trembleya parviflora,Tibouchina heteromalla and Eremanthus erythropappus showed Al concentrations higher than 1000 mg kg−1, which characterize them as hyperaccumulator species (Baker 1981Baker AJM. 1981. Accumulators and excluders: Strategies in the response of plants to heavy metals. Journal of Plant Nutrition 3: 643-654.).Lavoisiera pectinata, T. parviflora andT. heteromalla belong to Melastomataceae family, which comprises the highest number of Al-accumulator plant species (Jansen et al. 2002bJansen S, Watanabe T and Smets E. 2002b. Aluminium accumulation in leaves of 127 species in Melastomataceae, with comments on the order Myrtales. Ann Bot 90: 53-64.). The aluminium concentration found in L. clavatum in our study was higher than that observed on aerial parts of plants living in high Al-concentration soils byOlivares et al. (2009)Olivares E, Pena E, Marcano E, Mostacero J, Aguiar G, Benitez M and Rengifo E. 2009. Aluminium accumulation and its relationship whit mineral plant nutrients in 12 pteridophytes from Venezuela. Environ Exp Bot 65: 132-141.. According to Haridasan and Araújo (1988)Haridasan M and Araújo GM. 1988. A comparison of the nutrient status of two forests on dystrofhic and mesotrophic soils in the Cerrado region of central Brazil. Comm Soil Sci Plant Anal 19: 1075-1089., Eremanthus glomerulatus shows low aluminium concentration (150 mg kg−1), although other Asteraceae were described as hyperaccumulator species (Geoghegan and Sprent 1996Geoghegan IE and Sprent JL. 1996. Aluminium and nutrient concentrations in species native to central Brazil. Commun. Soil Sci Pl Anal 27: 2925-2934.). The low aluminium concentrations observed in Marcetia taxifolia, Nanuza plicata, Vellozia variegata, Myrsine umbellata and Baccharis trimera could be related to exclusion mechanisms or compartmentalization of this element in underground organs (Jansen et al. 2002aJansen S, Broadley MR, Robbrecht E and Smets E. 2002a. Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. Bot Rev 68: 235-269., Kochian et al. 2005Kochian LV, Pineros MA and Hoekenga OA. 2005. The physiology, genetics and molecular biology of plant aluminum resistance and toxicity. Plant Soil 274: 175-195., Hartwig et al. 2007Hartwig I, Oliveira AC, Carvalho FIF, Bertan I, Silva JAG, Schmidt DAM, Valério IP, Maia LC, Fonseca DNR and Reis CES. 2007. Mecanismos associados à tolerância ao alumínio em plantas. Semina Ciências Agrárias 28: 219-228.).

Some Al-accumulating plants releases organic acids to chelate Al3+ or maintain it within cells compartments, such as cell wall or vacuole (Taylor 1991Taylor GJ. 1991. Current views of the aluminum stress response: the physiological basis of tolerance. In: RANDALL DD, BLEVINS DG and MILES CD (Eds), Current Topics in Plant Biochemistry Physiology, Columbia: Missouri, v.10, p. 57-93., Delhaize and Ryan 1995Delhaize E and Ryan PR. 1995. Aluminum toxicity and tolerance in plants. Plant Physiol 107: 315-321., Shen et al. 2002Shen R, Ma J, Kyo M and Iwashita T. 2002. Compartmentation of aluminium in leaves of an Al-accumulator, Fagopyrum esculentum Moench. Planta 215: 394-398.). Among cell wall components, pectates are considered the main linkers to Al3+ (Chang et al. 1999Chang YC, Yamamoto Y and Matsumoto H. 1999. Accumulation of aluminum in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminum and iron. Plant Cell Environ 22: 1009-1017., Blamey 2001Blamey FPC. 2001. The role of the root cell wall in aluminum toxicity. In: AE N, ARIHARA J, OKADA K and SRINIVASAN A (Eds), Plant nutritent acquisition, Tokyo: Springer-Verlag, p. 201-226.). The histochemical tests indicated the primary cell walls as preferential sites of aluminium accumulation, possibly due to high content of pectates in relation to xylem secondary walls (Evert 2006Evert RF. 2006. Esau's Plant Anatomy, 3rd ed., New Jersey: Wiley-Interscience, 601 p.).

The intense color of the cuticle and bundle sheath cell walls inL. pectinata suggests a feasible mechanism whose function would be the protection of photosynthetic apparatus, as observed for Erica andevalensis by Turnau et al. (2007)Turnau K, Henriques FS, Anielska T, Renker C and Buscot F. 2007. Metal uptake and detoxification mechanisms in Erica andevalensis growing in a pyrite mine tailing. Environ Exp Bot 61: 117-123.. The positive response observed in chlorenchyma ofL. clavatum and T. parviflora suggests that chloroplasts could have an important role in accumulation of aluminium. High aluminium concentration observed in the mesophyll cells of pre-senescent leaves ofRicheria grandis is possibly stored in the vacuole and chloroplasts (Cuenca et al. 1991Cuenca G, Herrera R and Mérida T. 1991. Distribution of aluminium in accumulator plants by X-ray microanalysis in Richeria grandis Vahl leaves from a cloud forest in Venezuela. Plant Cell Environ 14: 437-441.).

Tests with Aluminon and Chrome Azurol represent techniques easily applied for preliminary studies, since they indicated, in this study, species with Al concentrations above 3000 mg kg−1. However, the histochemical tests are qualitative methods which are not able to discriminate between different Al concentrations. Aluminon is not a specific reagent for Al3+detection, and the intensity of its reaction can be influenced by other ions such as Fe3+ or Be2+, as well as other factors, such as cell type or intracellular pH (Clark and Krueger 1985Clark RA and Krueger GL. 1985. Review Article Aluminon: Its Limited Application as a Reagent for the Detection of Aluminum Species. J Histochem Cytochem 33: 729-732.). These limitations can explain the differences observed among the histochemical tests results.

Data obtained from pellets subjected to μ-EDX analyses were well correlated with the total Al concentrations obtained by ICP OES measurements, exhibiting linear correlation factor higher than 0.99, revealing that μ-EDX method is a suitable tool for Al quantification in plant samples.

The μ-EDX results obtained on dried leaves were consistent with those observed in histochemical tests, which showed a more homogeneous Al distribution in leaves of L. pectinata and L. clavatum. The low values obtained for Al in L. clavatum can be explained by a number of intercellular spaces presented in the leaf and stem of this specie, which can hamper the μ-EDX application due to its small spot size, 50 μm. The differences between the sampling points in T. parviflora may be related to higher cell density in leaf veins.

The feasibility of XRF technique on direct determination of elements in plant tissues was already demonstrated in several studies, such as Anjos et al. (2002)Anjos MJ, Lopes RT, Jesus EFO, Simabuco SM and Cesareo R. 2002. Quantitative determination of metals in radish using X-ray fluorescence spectrometry. X-Ray Spectrom 31: 120-123., Hokura et al. (2005)Hokura A, Onuma R, Kitajima N, Nakai I, Terada Y, Abe T, Saito H and Yoshida S. 2005. Cadmium Distribution in a Cadmium Hyperaccumulator Plant as Determined by Micro-XRF Imaging. Proc 8th Int Conf X-ray Microscopy, p. 323-325. and Marguí et al. (2009)Marguí E, Queralt I and Hidalgo M. 2009. Application of X-ray fluorescence spectrometry to determination and quantitation of metals in vegetal material. Trends Anal Chem 28: 362-372..

Despite all well documented limitations, mainly those related to matrix effects, the XRF technique is a fast and non-destructive method that can be successfully used in simultaneous determination of all target elements without requirement of laborious sample preparation steps (Saisho and Hashimoto 1996Saisho H and Hashimoto H. 1996. X-ray fluorescense analysis. In: SAISHO H AND GOHSHI Y (Eds), Applications of Synchontron Radiation to Materials Analysis. Amsterdam: Elsevier, p. 79-169., Torok et al. 1998Torok SB, Labar J, Schmeling M and Van Grieken RE. 1998. X-ray spectrometry. Anal Chem 70: 495-517.). The use of additional techniques, such as histochemical tests, can be used to complement data obtained from μ-EDX analysis.

CONCLUSIONS

Micro-energy dispersive X-ray fluorescence appears to be a suitable technique to Al quantification in pellets of ground plant tissues as well as for mapping studies in preserved dried leaves. Among the main advantages of this technique, we can mention (i) the low amount of sample required, (ii) its non-destructive capacity and (iii) achievement of reliable data without using expensive and hazardous chemicals.

Histochemical tests can be helpful for screening purposes of main bioaccumulation sites of samples before they are submitted to further μ-EDX scrutiny.

We would like to thank Bruno Tinti for helping us during sampling and for providing soil data. We wish to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2012/16203-5) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG 12070/2009) for financial support and grants.

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Publication Dates

  • Publication in this collection
    Mar 2014

History

  • Received
    13 July 2012
  • Accepted
    8 May 2013
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