The stage of seed development influences iron bioavailability in pea (Pisum sativum L.)

Table of Contents

Reagents

Chemicals, enzymes and hormones were purchased from Sigma-Aldrich, UK unless otherwise stated.

Plant material and growth

Pea seeds (Pisum sativum L.) representing a typical garden pea variety (genotype rr, accession number JI1194) were obtained from the John Innes Germplasm Resources Unit (www.jic.ac.uk/ germplasm/index.htm). Seeds were planted in peat-based compost plus grit, and grown in a greenhouse with partial temperature control. Plants were watered as required. Frozen garden peas were obtained from a local supermarket and dry mature pea seeds (Pisum sativum L. cv Sakura, marketed as marrowfat peas) were provided by Wherry & Sons (Bourne, UK).

Quantitative analysis of iron, phosphorus and phytic acid

The concentrations of iron and phosphorus were determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Fresh and mature pea seeds were dried at 55 °C overnight and ground to a fine powder with a mortar and pestle. Pea flour (50 mg) was acid digested in 2 ml nitric acid (69%) and 0.5 ml hydrogen peroxide (30%) for 6 h at 95 °C. The acid digests were diluted with 25 ml MilliQ water for ICP-OES. All analyses were carried out in triplicate from separate acid digestions. For measuring the iron concentration in simulated gastrointestinal digestions of cooked peas, 0.4 ml of digest was mixed with 0.4 ml nitric acid (69%) and 0.4 ml hydrogen peroxide (30%), and incubated at room temperature for 24 h and subsequently at 40 °C overnight. Samples were diluted with 4.32 ml MilliQ water to 5% nitric acid prior to ICP-OES.

The concentrations of phytic acid and total phosphorus were determined using a commercially available kit (K-PHYT 11/15 from Megazyme, Ireland).

In-gel iron staining

Pea ferritin was purified as previously described³⁰. For total protein extracts, pea flour or frozen pea samples were ground with a mortar and pestle in 10 volumes of 50 mM Tris-HCl pH 8.0, followed by centrifugation at 16,000 × g for 10 min to remove debris. Samples were kept at 4 °C throughout the protein extraction procedure to minimize degradation. The protein concentration was determined using BioRad Protein Assay Dye Reagent and bovine serum albumin as a standard. Purified ferritin (5–40 ng) or pea extract (20 µg protein) were mixed with loading buffer (20 mM Tris-HCl pH 8.0, 80% (v/v) glycerol, 0.1% (w/v) bromophenol blue) and separated on a non-denaturing gel of 6% (w/v) acrylamide:bis-acrylamide (37.5:1) in 0.375 M Tris-HCl pH 8.0 for 2 h at 30 mA. Duplicate gels were either stained for protein with Instant Blue (Expedeon) or for iron with enhanced Perls’/diaminobenzidine (DAB) staining³¹. For the latter, gels were incubated in 0.75% (w/v) HCl and 2% (w/v) ferrocyanide in H₂O for 20 min, then rinsed 4 × 5 min in MilliQ H₂O. Next, gels were incubated in 0.075% (w/v) DAB, 0.015% H₂O₂ for 20 min. Bands with a brown colour specific for Fe associated with ferritin started to appear within 10 min. When the colour was sufficiently developed, usually after 30 min, gels were rinsed in water (3 × 5 min). Gels were placed in ~10 ml water and kept at 4 °C for 2–3 days to further enhance the colour before digital imaging.

Immuno-detection of ferritin

Polyclonal antibodies against purified pea ferritin were raised in rabbit (Covalab, Cambridge, UK). The antisera were tested on known amounts of purified pea ferritin and on total proteins extracted from mature peas with 10% (w/v) trichloric acid in acetone. For most other purposes, proteins were extracted with 50 mM Tris-HCl pH 8.0. Protein extracts were mixed with loading buffer (0.125 M Tris-HCl pH 6.8, 2% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue, 5% (v/v) 2-mercaptoethanol) and separated on a 12.5% denaturing SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane using semi-dry blotting. Membranes were blocked in Tris-buffered saline (TBS), 1% (v/v) Tween-20 and 5% (w/v) skimmed milk (TBS-TM) for 1 h. Antibodies were diluted 1:5000 in TBS-TM and incubated for 1 h. Membranes were washed (3 × 5 min) with TBS-T, and then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Abcam UK, ab6721) at a 1:5000 dilution in TBS-T. After 4 × 5 min TBS-T washes, antibody binding was visualized with chemiluminescence reagents and digitally imaged using a LAS-500 fluorescent imager.

Immunolabelling of sections was as described³². The rabbit polyclonal antibodies against ferritin were detected using Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A11008). Images were acquired with a Zeiss Axiophot epifluorescence microscope using a Retiga EXT CCD digital camera (QImaging, Canada) and Metamorph software (Molecular Devices, USA).

Embedding and sectioning

Tissue samples of approximately 2 × 2 mm were infiltrated with 0.5 M MES-KOH pH 5.4, and high pressure frozen in 6 mm sample carriers using a high-pressure freezer (HPM 100 from Leica Microsystems, UK). Frozen samples were transferred to tubes containing frozen 100% (v/v) ethanol in liquid nitrogen and placed in an automatic freeze substitution system (EM AFS from Leica Microsystems, UK). Samples were sequentially warmed over 5 days to −30 °C, then to 4 °C over 48 h and finally to room temperature. The samples were processed through increasing concentrations of LR White resin (Agar Scientific UK, R1281) and embedded at 58 °C for 16–20 h in a nitrogen-rich environment. Sections (1 µm) of the resin blocks were cut with a Reichert-Jung ultramicrotome, and dried at 40 °C onto platinum-coated Thermanox coverslips for NanoSIMS, or on Polysine-coated slides (Agar Scientific UK, L4345) for immunolabelling.

NanoSIMS

NanoSIMS analysis was performed with a Cameca NanoSIMS 50 L (Cameca, France). A 16 keV Cs⁺ ion beam with a current of 1.5–2.5 pA and a beam size of approximately 150 nm (D1 = 2, 300 µm aperture) was focused onto the sample and rastered over the surface to generate negative secondary ions. The ions sputtered from the sample were analysed in a mass spectrometer to generate ion images of the tissue. Ion maps were simultaneously collected for ¹⁶O⁻, ¹²C¹⁴N⁻, ³²S⁻, ³¹P¹²C⁻, ³¹P¹⁶O⁻ and ⁵⁶Fe¹⁶O⁻ (all aligned on high concentration standards) as well as the secondary electron map. For each area a dose of 1 × 10¹⁷ Cs⁺ ions cm² was implanted before imaging by continuously scanning a large defocused beam to remove the platinum coating and maximize signal intensity. A 50 × 50 µm area was imaged with a dwell time of 5 ms per pixel. Sequential images from each region of interest were acquired and summed to improve the counting statistics. The number of images acquired was varied to give the same total dose for each region depending on the beam current. Image processing was conducted with ImageJ using the OpenMIMS plugin (Harvard).

In vitro iron bioavailability studies

Garden peas were microwaved following the cooking instruction on the pack. In brief, 100 g frozen garden peas were placed in a beaker with 1 tablespoon (9.5 ml) MilliQ water (18.2 MΩ), and microwaved at 900 W for 2.5 min. Dried peas (100 g) were soaked in 500 ml Milli-Q water overnight, rinsed 3 times and boiled with 400 ml MilliQ water for 1 h. After cooking, samples were frozen at −80 °C, lyophilized, finely ground and stored at 4 °C until further use.

The simulated gastrointestinal digestion was performed as described¹⁹ with minor modifications. Pea samples were added to 10 ml of saline solution (140 mM NaCl, 5 mM KCl) at pH 2.0, followed by addition of pepsin (0.04 g ml⁻¹) and incubated for 90 min on a rolling platform at 37 °C to simulate gastric conditions. Ascorbic acid was added at a molar ratio of 10:1 ascorbate:iron. Subsequently, the pH of the samples was gradually adjusted to pH 5.5 with NaHCO₃. Bile extract (0.007 g ml⁻¹) and pancreatin (0.001 g ml⁻¹) were added, samples were readjusted to pH 7, and incubated for an additional hour on a rolling platform at 37 °C to mimic intestinal conditions. At the end of the simulated gastrointestinal digestion, samples were centrifuged at 3000 × g for 10 min and the supernatants were used for subsequent cell culture experiments. A volume of 1.5 ml supernatant was applied to an upper chamber consisting of a Transwell insert fitted with a 15 kDa molecular weight cut-off dialysis membrane (Spectra/Por 7 dialysis tubing, Spectrum Laboratories, Europe) suspended over Caco-2 cell monolayers grown in collagen-coated 6-well plates. After incubation for one hour at 37 °C in a humidified incubator containing 5% CO₂ and 95% air, inserts were removed and an additional 1 ml of low-iron Eagle’s minimal essential medium (MEM, GIBCO, Grand Island, NY) was added. Cells were incubated for a further 23 hours prior to harvesting for ferritin analysis. Details of Caco-2 cell culture and ferritin analysis by ELISA are described in ref.³³. To assess the effect of phytic acid on iron bioavailability, either 0.5 mM phytic acid was added to the digestate of microwaved garden peas, or 480 U phytase enzyme (Megazyme) was added to the digestate of boiled mature peas.

Statistical analysis

Quantitative data are presented as mean values with standard errors of the means (SEM). Homogeneity of variance was tested using the Levene’s test. For multiple comparison, one-way ANOVA was used followed by a post-hoc Tamhane test for non-homogenous variances. The Student’s t-test was used when two treatments were compared to each other. Statistical significance was set at p < 0.05. The statistical analysis was performed using the SPSS package (version 23; SPSS Inc., Chicago, IL, USA).