Selenium accumulation, distribution, and speciation in spineless prickly pear cactus: a drought- and salt-tolerant, selenium-enriched nutraceutical fruit crop for biofortified foods

Abstract

The organ-specific accumulation, spatial distribution, and chemical speciation of selenium (Se) were previously unknown for any species of cactus. We investigated Se in Opuntia ficus-indica using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (μXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). μXRF showed Se concentrated inside small conic, vestigial leaves (cladode tips), the cladode vasculature, and the seed embryos. Se K-edge XANES demonstrated that approximately 96% of total Se in cladode, fruit juice, fruit pulp, and seed is carbon-Se-carbon (C-Se-C). Micro and bulk XANES analysis showed that cladode tips contained both selenate and C-Se-C forms. Inductively coupled plasma mass spectrometry quantification of Se in high-performance liquid chromatography fractions followed by LC-MS structural identification showed selenocystathionine-to-selenomethionine (SeMet) ratios of 75:25, 71:29, and 32:68, respectively in cladode, fruit, and seed. Enzymatic digestions and subsequent analysis confirmed that Se was mainly present in a "free" nonproteinaceous form inside cladode and fruit, while in the seed, Se was incorporated into proteins associated with lipids. μXRF chemical mapping illuminated the specific location of Se reduction and assimilation from selenate accumulated in the cladode tips into the two LC-MS-identified C-Se-C forms before they were transported into the cladode mesophyll. We conclude that Opuntia is a secondary Se-accumulating plant whose fruit and cladode contain mostly free selenocystathionine and SeMet, while seeds contain mainly SeMet in protein. When eaten, the organic Se forms in Opuntia fruit, cladode, and seed may improve health, increase Se mineral nutrition, and help prevent multiple human cancers.

Figure 1.

Photographs showing the organs and tissues of spineless, salt- and B-tolerant, dark purple fruit-bearing Opuntia (USDA No. 248) subjected to biochemical analysis. Young cladode with tips (A), fruit (B), and seeds (C) are shown.

Figure 2.

Distribution and speciation of Se in Opuntia. μXRF map showing spatial distribution of Se (coded in red) and other selected elements (calcium [Ca] in green, potassium [K] or zinc [Zn] in blue) in cladodes (A and C), in seeds (B), and in fruit (D). Bars = 600 μm (A), 1 mm (B), and 900 μm (C and D). Yellow circles in A to C show locations of μXANES scans whose fitting results are reported in Table I.

Figure 3.

A, Normalized Se K-edge XANES spectra of different Opuntia samples. Reference spectra of SeMet, trimethylselenonium ion [(CH₃)₃Se⁺], and SeO₄²⁻ are shown for comparison. The SeO₄²⁻ spectrum is shown as a dashed line. The dotted line indicates the peak position of SeMet standard. B, Distribution of Se in Opuntia by μXRF map showing the spatial distribution of Se (coded in white) in young cladode. Bar = 1 mm.

Figure 4.

Se-containing fractions of Opuntia cladode (A), clarified fruit juice (B), fruit pulp (C), and seed (D). Samples were predigested with proteinase K (black circles) or not digested (white circles). Values were quantified in nL L⁻¹ (ppb) and obtained by reverse-phase C18 HPLC followed by ICPMS.

Figure 5.

LC-MS of 1.7- to 2.0-min Se-containing fractions with a mass spectrum matching SeCyst in cladode (A) and fruit juice (B). The published mass spectrum for SeCyst is shown in C (Dernovics et al., 2007; Freeman et al., 2010).

Figure 6.

LC-MS of 3.0- to 3.3-min Se-containing fraction with a mass spectrum and retention time matching SeMet in cladode (A), fruit juice (B), and seed (C). The mass spectrum and retention time for a SeMet standard are shown in D.

Figure 7.

A, μXRF map showing spatial distribution of Se (coded in red) and other selected elements (calcium [Ca] in green, potassium [K] in blue) in a young cladode from a plant supplied with 50 μm Na₂SeO₄. B, Bicolor-coded chemical map of a young cladode showing SeO₄²⁻ in red and C-Se-C (SeMet or SeCyst) in green, using 35- × 35-μm² pixel size. Bars = 1 mm.