Using μPIXE for quantitative mapping of metal concentration in Arabidopsis thaliana seeds

Abstract

Seeds are a crucial stage in plant life. They contain the nutrients necessary to initiate the development of a new organism. Seeds also represent an important source of nutrient for human beings. Iron (Fe) and zinc (Zn) deficiencies affect over a billion people worldwide. It is therefore important to understand how these essential metals are stored in seeds. In this work, Particle-Induced X-ray Emission with the use of a focused ion beam (μPIXE) has been used to map and quantify essential metals in Arabidopsis seeds. In agreement with Synchrotron radiation X-ray fluorescence (SXRF) imaging and Perls/DAB staining, μPIXE maps confirmed the specific pattern of Fe and Mn localization in the endodermal and subepidermal cell layers in dry seeds, respectively. Moreover, μPIXE allows absolute quantification revealing that the Fe concentration in the endodermal cell layer reaches ~800 μg·g(-1) dry weight. Nevertheless, this cell layer accounts only for about half of Fe stores in dry seeds. Comparison between Arabidopsis wild type (WT) and mutant seeds impaired in Fe vacuolar storage (vit1-1) or release (nramp3nramp4) confirmed the strongly altered Fe localization pattern in vit1-1, whereas no alteration could be detected in nramp3nramp4 dry seeds. Imaging of imbibed seeds indicates a dynamic localization of metals as Fe and Zn concentrations increase in the subepidermal cell layer of cotyledons after imbibition. The complementarities between μPIXE and other approaches as well as the importance of being able to quantify the patterns for the interpretation of mutant phenotypes are discussed.

Keywords: Arabidopsis; elemental mapping; iron; quantitative; seed; μPIXE.

Figure 1

Arabidopsis thaliana dry seed structure. Light microscopy images of transversal (A) and longitudinal (B) sections with respective schematic representations (C,D). Thirty micrometer sections were obtained at −30°C using a cryomicrotome. Scale bar: 100 μm; asm, apical shoot meristem; cot, cotyledon; end, endodermis; hyp, hypocotyl; rad, radicle; sc, seed coat.

Figure 2

μPIXE elemental reconstituted image of Arabidopsis thaliana dry seed section. Total X-ray spectra of areas scanned with 3 MeV proton beam were collected. Specific distributions of Fe, Mn, and Ca in a 30 μm thick longitudinal section of Arabidopsis dry seed are shown overlaid (large image) and separately (small images). False color images and reconstitution were obtained using ImageJ free software. Scale bar: 100 μm; red, Fe; blue, Mn; yellow, Ca.

Figure 3

Regions of interest for μPIXE elemental quantification. Element distribution maps allowed selection of regions of interest (ROI). Arabidopsis thaliana whole seed (A), Fe- (B), and Mn- (C) ROIs are shown using the Ca distribution map as background. The associated STIM image (D) shows the local mass of the sample which is necessary for quantification. Scale bar: 100 μm.

Figure 4

Transversal sections of Arabidopsis thaliana dry and imbibed seeds. Specific distributions of Fe, Mn, and Ca in section of WT (A) and nr3nr4 (B) imbibed seeds and vit1-1 (C) dry seeds are shown overlaid (large image) and separately (small images). Scale bar: 100 μm; red, Fe; blue, Mn; yellow, Ca.

Figure 5

Metal content in Arabidopsis thaliana dry (A) and imbibed (B) seed. Fe, Mn, and Zn contents in whole seed section (black bars) as well as in selected Fe- (white bars) and Mn- (gray bars) enriched areas were quantified. Dry seeds were cryofixed directly (A) or after 48 h imbibition (B). Values are means ± SE (n = 3–4 scanned sections). Different letters denote statistically significant differences based on Kruskal–Wallis test followed by Tukey post-hoc test comparing values of dry and imbibed seeds for each metal (P < 0.05).

Figure A1

μPIXE elemental reconstituted image of Arabidopsis thaliana dry seed section. Total X-ray spectra of areas scanned with 3 MeV proton beam were collected. Specific distributions of Ca, K, Fe, Mn, and Zn in a 30 μm thick longitudinal section of Arabidopsis dry seed are separately. The associated STIM image shows the local mass of the sample. Scale bar: 100 μm.