Contents lists available at ScienceDirect Spectrochimica Acta Part B journal homepage: www.elsevier.com/locate/sab The use of micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) combined with a multivariate approach to determine element variation and distribution in tobacco seedlings exposed to arsenate G. Capobianco a , P. Brunetti b , G. Bonifazi a , P. Costantino b,c , M. Cardarelli b, ⁎ , S. Serranti a, ⁎ a Dip. Ingegneria Chimica Materiali Ambiente, Sapienza Università di Roma, Italy b IBPM-CNR c/o Dip. Biologia e Biotecnologie, Sapienza Università di Roma, Italy c Dip Biologia e Biotecnologia, Sapienza Università di Roma ARTICLE INFO Keywords: X-ray fluorescence spectrometry Multivariate approach Nicotiana tabacum seedlings Arsenic distribution Elemental map ABSTRACT Here, we present a new scheme of analysis combining micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) with a multivariate approach that allows to establish the inter-correlation of multiple elements and their elemental map in plants. The main advantage of this procedure is that XRF spectral profiles can be analysed directly, by means of principal component analysis (PCA), allowing a quick interpretation of the results. Furthermore, this analysis requires small amounts of plant material and can be performed in whole individual seedlings in the hydrated state without chemical extraction. With this technology, we determined the dis- tribution of arsenic (As) and the variation and spatial distribution of multiple elements in whole tobacco seedlings grown in the presence of different arsenate concentrations. We observed that As is detectable mainly in roots, primarily in the basal part, but also in the root apex in seedlings grown in the highest arsenate con- centration. The low rate of As translocation from roots to shoots and the significant increase in S are consistent with previous evidence showing that As is retained in roots by forming complexes with thiol peptides. We also found a significant non-linear increase in P, as arsenate is taken up by phosphate transporters and induces the expression of genes encoding them. A decrease in Mn, Fe and Zn proportional to the accumulation of As and in the same tissues, suggests a competition of these elements with As for cellular transporters. 1. Introduction Micro X-ray fluorescence (μ-XRF) is a valuable and sensitive tool for the analysis of the distribution of elements in different regions of a plant. In recent years this technique has been used in combination with or has often replaced conventional approaches. The quantitative de- termination of multiple elements in plants has been generally per- formed by acid digestion of the samples followed by ICP-OES analysis (inductively coupled plasma-optical emission spectrometer) [1,2]. This approach requires large amounts of plant material and is therefore performed on a limited number of samples from removed plants and it does not give any information about the topological distribution of elements. Analyses of plant tissues by synchrotron X-ray fluorescence techniques indeed drastically reduced the amount of required material but mainly dehydrated tissues are utilized [3]. Since X-rays can be fo- cused to spots smaller than 1 mm, researchers have used it to identify and quantify nutrients and non-essential elements such as arsenic (As) or cadmium (Cd) [4]. Synchrotron-based X-ray microfluorescence imaging analysis was applied to characterize the simultaneous sub- cellular distribution of a number of mineral elements. This fine-imaging method can reveal whether these elements colocalize [5–6]. Very re- cently a μ-XRF instrument has been designed and applied for in vivo analysis of Arabidopsis leaves- not detached from the plant- aiming to reduce as much as possible damages to the plant tissues [7]. μ-XRF technique has proved particularly effective in the study of elemental distribution in different organs of plants exposed to different elemental contaminants [8,9,10]. The most frequent contaminants of soils are heavy metals, including As, and among them As is recognized as one of the main toxicants worldwide. Arsenic is released into the environment both by natural processes, such as weathering of rocks, and by anthropogenic activities such as mining or the use of As-containing pesticides, herbicides and wood preservatives [11]. The accumulation of As in the biosphere and in the food chain has serious effects on the environment and human health [12]. Plants have widely different capabilities of tolerating and https://doi.org/10.1016/j.sab.2018.05.029 Received 21 September 2017; Received in revised form 28 May 2018; Accepted 30 May 2018 ⁎ Corresponding authors. E-mail addresses: maura.cardarelli@uniroma1.it (M. Cardarelli), silvia.serranti@uniroma1.it (S. Serranti). Spectrochimica Acta Part B 147 (2018) 132–140 Available online 30 May 2018 0584-8547/ © 2018 Published by Elsevier B.V. T