Comparison of Zn accumulation and speciation in kernels of sweetcorn and maize differing in maturity

Abstract

Background and Aims

Understanding the speciation of Zn in edible portions of crops helps identify the most effective biofortification strategies to increase the supply of nutrients for improving the health and nutrition of consumers.

Methods

Kernels of 12 sweetcorn and three maize (Zea mays) varieties were analysed for Zn concentration and content. The speciation of the Zn in the embryos, endosperms and whole kernels at 21, 28 and 56 days after pollination (DAP) was then examined for one maize and one sweetcorn variety using synchrotron-based X-ray absorption spectroscopy (XAS).

Key Results

Averaged across all sweetcorn and maize varieties at 21 DAP, the embryo contributed 27–29% of the whole kernel Zn whilst the endosperm contributed 71–73 %. While sweetcorn embryos contributed a lower proportion to the total kernel Zn than those of maize, the proportion of total Zn in the embryo increased as kernels aged for both varieties, reaching 33 % for sweetcorn and 49% for maize at 28 DAP. Using XAS, it was predicted that an average of 90 % of the Zn in the embryos was present as Zn-phytate, while in the endosperm the Zn was primarily complexed with an N-containing ligand such as histidine and to a lesser extent with phytate. However, in maize endosperms, it was also observed that the proportion of Zn present as Zn-phytate increased as the kernel matured, thereby also probably decreasing its bioavailability in these mature maize kernels.

Conclusions

The apparent low bioavailability of Zn supplied in maize at its consumption stage (i.e. mature kernels) probably undermines the effectiveness of biofortification of this crop. Conversely, successful biofortification of Zn in sweetcorn and green maize consumed as immature kernels could potentially provide a good source of bioavailable Zn in human diets.

Introduction

Zinc (Zn) is an essential micronutrient for humans, animals and plants, having roles in enzyme function, protein synthesis and in the immune system (Coleman, 1992; Andreini et al., 2006; Maret and Sandstead, 2006). It is estimated that half of the world’s agricultural lands are Zn deficient (Alloway, 2008), with approx. 30 % of the global human population having an inadequate dietary intake of Zn (Welch and Graham, 2004; Black et al., 2013). Biofortification aims to increase bioavailable concentrations of essential nutrients, such as Zn, in edible portions of crop plants through genetic selection or agronomic intervention (White and Broadley, 2005).

Successful biofortification of food crops aims to increase not only the nutrient content and concentration delivered in a diet, but also the bioavailability of these nutrients to consumers after consumption. The bioavailability of micronutrients is influenced by their speciation within the plant tissues and within the gastrointestinal tract after consumption (Graham et al., 2000; Welch and Graham, 2004). For example, micronutrients in cereal grains are often complexed with phytate (Hunt, 2003; Raboy, 2007; Bohn et al., 2008) and, given that the human gut lacks the phytase enzyme, these micronutrients are therefore not absorbed (Iqbal et al., 1994; Lönnerdal, 2002). In contrast, the presence of promoters, such as lysine, β-carotene, vitamin A and vitamin C, could enhance the bioavailability of micronutrients (Garcia-Casal et al., 1998; Hambidge et al., 2007).

The bioavailability of Zn in maize (Zea mays) is low due to the high phytate concentration in its kernels (Schlemmer et al., 2009; Gibson et al., 2010). However, evidence suggests that the majority of this phytate content may accumulate between 15 and 30 days after pollination (DAP) (Raboy et al., 2000). Furthermore, there is recent evidence that while the Zn in the embryo of sweetcorn (Zea mays L. ssp. saccharata) is mainly associated with phytate, the Zn in the endosperm is associated with nitrogen (N)- or sulfur (S)-containing ligands (Cheah et al., 2019a). This is consistent with earlier reports that Zn in various other cereal grains is often complexed with proteins or other ligands instead of phytate (Lee et al., 2011; Hansen et al., 2012; Velu et al., 2014; Persson et al., 2016). These results suggest that the bioavailability of Zn in maize and sweetcorn kernels could be higher than previously estimated, and potentially able to deliver better outcomes for biofortification efforts.

The aim of the present study was to examine the accumulation and speciation of Zn within sweetcorn and maize kernels at different stages of maturity. First, we determined the Zn concentration and content of embryo and endosperm tissues in 12 varieties of sweetcorn and three varieties of maize at the immature stage (21 DAP) corresponding to the time of sweetcorn consumption. Next, using one variety of sweetcorn and one variety of maize, we examined the change in the Zn concentration and content within these two tissues of sweetcorn and maize over five different stages of kernel maturity. Finally, we used synchrotron-based X-ray absorption spectroscopy (XAS) for the in situ examination of the speciation of Zn within the embryo and endosperm. This information is critical in understanding how these two major crops contribute to the dietary Zn nutrition of consumers.

MATERIALS AND METHODS

Plant material

Twelve sweetcorn and three maize varieties (Table 1) were grown in the field at the Gatton Research Facility of the Queensland Department of Agriculture and Fisheries (Gatton, Australia) on a Vertisol soil (Soil Survey Staff, 2014). A pre-planting basal fertilizer delivering an elemental equivalent of 55 kg N ha^–1, 10 kg P ha^–1, 55 kg K ha^–1 and 80 kg S ha^–1 was applied. Additional fertilizers were applied using trickle irrigation, being a urea–NH₄–NO₃ mix (Easy-N^®) at a rate of 13.5 kg N ha^–1, and a further application of 26 kg N ha^–1, 33 kg S ha^–1 and 2 kg Mg ha^–1 as ammonium sulfate [(NH₄)₂SO₄] and magnesium sulfate (MgSO₄). Finally, zinc oxide (ZnO) was applied once at the 12-leaf stage at a rate of 0.6 kg Zn ha^–1 as a foliar application. During the growth period, the average temperature range was 15–30 °C and average relative humidity range was 44–59 %.

Table 1.

Means and standard errors of tissue Zn concentration and the mass of Zn in embryos, endosperms and whole kernels from 15 sweetcorn and maize (Zea mays) varieties sampled 21 days after pollination

Variety	Zn concentration (mg kg–1 d. wt)			Zn content (µg per kernel)			Proportion of total kernel Zn (and dry weight) (%)
	Whole kernel	Embryo	Endosperm	Whole kernel	Embryo	Endosperm	Embryo	Endosperm
Sweetcorn
6-1 × 15-2	28 ± 1.1	121 ± 6.4	22 ± 0.9	3.5 ± 0.4	0.9 ± 0.2	2.6 ± 0.2	27 (6)	73 (94)
Hybrix 5	28 ± 2.9	113 ± 4.9	23 ± 1.4	3.5 ± 0.2	0.8 ± 0.2	2.7 ± 0.1	23 (5)	77 (95)
23-1	33 ± 2.6	128 ± 18.8	26 ± 2.3	3.4 ± 0.4	0.9 ± 0.1	2.5 ± 0.3	28 (8)	72 (92)
HiZeax 103146	30 ± 0.6	132 ± 5.1	21 ± 0.4	3.4 ± 0.1	1.1 ± 0.1	2.2 ± 0.1	34 (8)	66 (92)
56.3-1	41 ± 3.3	167 ± 17.5	28 ± 2.6	3.3 ± 0.4	1.3 ± 0.1	2.1 ± 0.3	38 (10)	62 (90)
14–6	44 ± 6.4	168 ± 23.7	39 ± 6.7	2.9 ± 0.5	0.5 ± 0.2	2.4 ± 0.4	17 (5)	83 (95)
13-2	33 ± 2.0	182 ± 12.8	24 ± 2.0	2.9 ± 0.2	1.0 ± 0.1	1.9 ± 0.1	33 (6)	67 (94)
O2su	30 ± 2.9	156 ± 34.5	26 ± 1.4	2.7 ± 0.3	0.5 ± 0.1	2.2 ± 0.2	19 (4)	81 (96)
23–7	30 ± 0.4	132 ± 9.4	22 ± 0.5	2.5 ± 0.2	0.8 ± 0.1	1.7 ± 0.1	33 (8)	67 (92)
23–6	29 ± 1.6	104 ± 5.7	22 ± 1.7	2.3 ± 0.2	0.7 ± 0.1	1.6 ± 0.1	31 (8)	69 (92)
Garrison	20 ± 1.0	55 ± 2.3	17 ± 1.0	2.3 ± 0.2	0.5 ± 0.0	1.7 ± 0.1	23 (8)	77 (92)
fl2sh2	25 ± 1.7	99 ± 22.1	23 ± 1.9	1.4 ± 0.3	0.2 ± 0.1	1.2 ± 0.2	17 (4)	83 (96)
Mean	31 ± 2.2	130 ± 13.6	24 ± 1.9	2.8 ± 0.3	0.8 ± 0.1	2.1 ± 0.2	27 (7)	73 (93)
Maize
Dull purple	20 ± 0.6	86 ± 7.7	15 ± 0.7	2.9 ± 0.2	0.8 ± 0.1	2.1 ± 0.1	27 (6)	73 (94)
Thai Floury 2	28 ± 2.4	130 ± 7.7	20 ± 0.8	2.9 ± 0.2	0.9 ± 0.0	2.0 ± 0.1	31 (7)	69 (93)
EM 540	26 ± 1.1	136 ± 12.5	20 ± 1.0	2.7 ± 0.1	0.8 ± 0.1	1.9 ± 0.1	30 (6)	70 (94)
Mean	25 ± 1.4	117 ± 9.3	18 ± 0.8	2.8 ± 0.2	0.8 ± 0.1	2.0 ± 0.1	29 (6)	71 (94)

The proportion of the total kernel Zn (and dry matter in parentheses) found in the embryo and endosperm is also shown. Concentration data are reported on a dry weight basis.

All varieties were manually pollinated before being harvested at 21 DAP. Additionally, the ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize varieties were also harvested at 18, 24, 28 and 56 DAP. The 21 DAP stage has immature kernels that corresponds to the timing of early commercial sweetcorn harvesting, 24 DAP corresponds to late commercial sweetcorn harvesting and 56 DAP corresponds to kernel maturity when maize is harvested. Harvested cobs with immature kernels (i.e. 18–28 DAP) were transported to an air-conditioned laboratory where a portion of the kernels was immediately excised from the cobs and dissected into the embryo and endosperm tissues. We were unable to dissect kernels at 56 DAP into the respective tissues as the plant material was too dry and hard. After dissection, all samples were immediately frozen in liquid nitrogen before being freeze-dried. Other sub-samples of the kernels, which were not dissected, were frozen in liquid nitrogen intact before being freeze-dried. Dry weight measurements of kernels and the respective tissue components were taken.

Bulk tissue analyses

Zinc concentrations were determined for bulk (whole) kernels as well as for the dissected embryo and endosperm tissues. Samples of the freeze-dried whole kernels and the respective tissues were digested by adding a mixture of 6 mL of nitric acid and 4 mL of perchloric acid, and heated to 150 °C. When the mixture turned clear indicating that all organic material has been digested, deionized water was added to make up the solution to 20 mL. The digested samples were analysed using inductively coupled plasma optical emission spectroscopy (ICP-OES) (Optima 7300 DV, Perkin Elmer; Wellesley, MA, USA) with a detection limit of 0.2 mg Zn L^–1. A 1000 mg Zn L^–1 solution (Australian Chemical Reagents, South Australia) was used as the Zn standard, while various plant materials (Global Proficiency, Hamilton, New Zealand) were used as certified reference materials. The Zn contents of the bulk kernels and various tissues were calculated from the dry weight.

X-ray absorption near-edge structure (XANES) analysis

The speciation of Zn within the whole kernel, embryo and endosperm tissues was examined for two representative varieties, namely ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize. ‘Hybrix 5’ is a commercially cultivated sweetcorn variety in Australia, while ‘Thai Floury 2’ is a quality protein maize variety developed for protein biofortification. Samples were examined for both varieties at 21, 28 and 56 DAP, with in situ analyses conducted using synchrotron-based XAS at Sector 10-ID (Segre et al., 2000) of the Advanced Photon Source (Lemont, IL, USA). The energy of each spectrum was calibrated by the simultaneous measurement of a Zn foil in transmission. The data were collected in fluorescence modes using a Lytle detector. The size of the incident X-ray beam was approx. 0.5 × 0.5 mm.

Prior to scanning, freeze-dried kernel tissue samples were homogenized using a stainless steel grinder and an agate mortar and pestle. After grinding, the samples were pressed into 7 mm pellets, sealed with Kapton tape and transferred to a sample holder. We examined a total of 14 samples, which consist of embryo tissues at 21 and 28 DAP, endosperm tissues at 21 and 28 DAP, and whole kernel samples at 21, 28 and 56 DAP for both ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize. Eight replicate scans were conducted on each sample, with each individual scan being conducted at a new location on the sample pellet.

In addition to the 14 kernel samples, a total of nine standard compounds were scanned. For four of these nine standards, solutions were prepared by mixing ZnSO₄·7H₂O with the appropriate ligand (citric acid, phytic acid, histidine or cysteine) to achieve a final Zn concentration of 20 mm and a final ligand concentration of 100 mm. Solutions were adjusted to pH 7 using NaOH. The solutions were then frozen in liquid nitrogen and freeze-dried to obtain a powder. These four standards, together with four other solid standards [ZnCO₃, ZnO, Zn(OH)₂ or Zn₂(PO₄)₃] were diluted to 150 mg g^–1 using cellulose. The metallic Zn reference foil was used as the final standard. For each of these nine standard compounds, four replicate scans were conducted.

The XANES spectrum for each scan was normalized using the reference energy from the Zn foil measured in transmission. Replicate scans were averaged in Athena v.0.8.056 (Ravel and Newville, 2005). Data were processed using linear combination fitting (LCF) with a fitting range of –20 to +30 eV relative to the Zn K-edge. The LCF was used to identify the relative proportions of the various standard spectra within each sample spectrum. A maximum of three standard compounds were allowed to be fitted for each sample. Tissue concentrations were too low to permit the collection of the extended X-ray absorption fine-structure (EXAFS) spectra.

X-ray fluorescence microscopy (XFM) analysis

The distribution of a range of elements, including Zn, P and S, was examined in situ using the immature (21 DAP) and mature (56 DAP) kernels for the ‘Hybrix 5’ sweetcorn variety. Analyses were conducted using the XFM beamline at the Australian Synchrotron (Melbourne, Australia). The sample preparation and scanning methodology have been previously described (Cheah et al., 2019b). Briefly, cut and polished kernel sections were attached on Perspex^® sample mounts using double-sided tape. A monochromatic beam, 12.9 keV, was focused using Kirkpatrick–Baez mirrors to approx. 2 × 2 µm. A Vortex-EM silicon drift detector was placed at 90° to the incident beam and processed using a FalconX (XIA) digital signal processor. Detailed elemental mapping was conducted with discrete steps in the vertical direction and on-the-fly scanning in the horizontal direction. Scans were conducted with 10 μm sampling step size intervals at a sample stage speed of 1 mm s^–1 and a transit time per image pixel of approx. 10 ms (4 μm intervals, 0.5 mm s^–1 speed and 8 ms transit time for embryo scans). The CSIRO Dynamic Analysis method in GeoPIXE provided quantitative, true-element images of X-ray fluorescence spectra (Ryan, 2000).

RESULTS

Tissue Zn concentration and content analysis: variety comparison

At 21 DAP, the average whole kernel Zn concentration for sweetcorn varieties was 24 % higher than that in maize (Table 1), although there was a wide variation in Zn concentration among sweetcorn varieties (20–44 mg kg^–1) compared with the maize varieties (20–28 mg kg^–1). Importantly, averaged across all sweetcorn and maize varieties, it was found that Zn concentrations were 5.5 times higher in the embryo (average of 127 mg kg^–1) than in the endosperm (average of 23 mg kg^–1). As found for the whole kernels, Zn concentrations in the endosperm at 21 DAP were higher (33 %) for sweetcorn than for maize, and embryo Zn concentrations were also higher (11 %) in sweetcorn than in maize.

However, despite the tendency for higher Zn concentrations in sweetcorn genotypes, differences in kernel dry weight resulted in the average kernel Zn content being comparable in both sweetcorn and maize (2.8 µg Zn per kernel; Table 1). This similarity held for both embryo and endosperm components (0.8 µg Zn per embryo and 2.0–2.1 µg Zn per endosperm). This observation was consistent with the typically higher dry matter content of maize kernels (34–51 %) at this stage of maturity as compared with sweetcorn kernels (23–32 %, unpubl. data.)

The markedly higher Zn concentrations in the embryo tissue of both maize and sweetcorn resulted in embryos contributing a much higher proportion to the total kernel Zn (27 % for sweetcorn and 29 % for maize) than would have been expected on the basis of the proportion of those tissues on a dry weight basis (6–7 % of kernel dry weight in sweetcorn and maize; Table 1). These data also highlight the importance of the endosperm as a key storage organ for Zn accumulation at this stage of kernel development (approx. 71–73 % of the total kernel Zn content), which coincides with the sweetcorn consumption stage.

Influence of maturity on Zn concentration and content

The influence of kernel maturity on Zn concentration and content was explored using the commercial sweetcorn variety ‘Hybrix 5’ and the quality protein maize variety ‘Thai Floury 2’. In the sweetcorn variety ‘Hybrix 5’, Zn concentration in both the embryo and endosperm tissues rapidly decreased between 18 and 21 DAP, before remaining similar thereafter (Table 2). Although the endosperm concentration at 24 DAP was significantly different from those at 21 and 28 DAP (in contrast to embryo concentrations), the magnitude of the difference was not substantial. Given that the endosperm constitutes a larger proportion of the total dry weight, changes in the whole kernel Zn concentration most closely resembled those of the endosperm, decreasing from 37 mg kg^–1 at 18 DAP to 28 mg kg^–1 at 21 DAP, then fluctuating between 28 and 33 mg kg^–1 thereafter (Table 2). These results suggest that from 18 to 21 DAP, dry matter accumulation was faster than Zn accumulation, whereas, from 21 DAP onwards, both Zn and dry matter accumulated in the kernel at relatively constant proportions.

Table 2.

Means and standard errors of tissue Zn concentration and the mass of Zn in embryos, endosperms and whole kernels of ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize (Zea mays) at 18, 21, 24, 28 and 56 days after pollination (DAP)

Tissue	Zn concentration (mg kg–1 d. wt)					Zn content (µg per kernel)
	18 DAP	21 DAP	24 DAP	28 DAP	56 DAP	18 DAP	21 DAP	24 DAP	28 DAP	56 DAP
‘Hybrix 5’ sweetcorn
Embryo	138 ± 2.6a	113 ± 4.9b	112 ± 3.5b	104 ± 7.4b	–	0.5 ± 0.1	0.8 ± 0.2	1.5 ± 0.1	1.9 ± 0.1	–
Endosperm	32 ± 1.6a	23 ± 1.4c	26 ± 0.9b	23 ± 1.5c	–	2.1 ± 0.1	2.7 ± 0.1	3.7 ± 0.3	3.9 ± 0.3	–
Whole kernel	37 ± 1.2a	28 ± 2.9c	33 ± 0.7b	31 ± 2.0bc	32 ± 1.8b	2.6 ± 0.2	3.5 ± 0.2	5.2 ± 0.3	5.8 ± 0.4	8.8 ± 0.7
Embryo (%)						18	23	28	33
Endosperm (%)						82	77	72	67
‘Thai Floury 2’ maize
Embryo	112 ± 8.0c	130 ± 7.7b	145 ± 8.0a	92 ± 9.1d	–	0.5 ± 0.1	0.9 ± 0.0	2.3 ± 0.4	3.3 ± 0.2	–
Endosperm	18 ± 1.9a	20 ± 0.8a	21 ± 1.8a	14 ± 0.6b	–	1.3 ± 0.1	2.0 ± 0.1	2.9 ± 0.2	3.4 ± 0.0	–
Whole kernel	24 ± 1.2c	28 ± 2.4b	34 ± 1.8a	25 ± 2.3bc	23 ± 1.1c	1.8 ± 0.2	2.9 ± 0.2	5.2 ± 0.2	6.7 ± 0.3	10.5 ± 0.6
Embryo (%)						28	31	44	49
Endosperm (%)						72	69	56	51

Concentration data are reported on a dry weight (d. wt) basis. Means that do not share the same letter indicate significant differences within tissue types at various stages of maturity.

In contrast to the endosperm, embryo Zn concentration of the maize variety ‘Thai Floury 2’ increased from 18 to 24 DAP, before decreasing rapidly between 24 and 28 DAP (Table 2). Meanwhile, the endosperm remained at similar concentrations from 18 to 24 DAP before also decreasing rapidly between 24 and 28 DAP. Therefore, the whole kernel Zn concentration was highest at 24 DAP (34 mg kg^–1), declining before and after this maturity stage. These results suggest that in maize, the milk stage (i.e. 21–24 DAP) corresponded to rapid kernel Zn accumulation, followed by a major influx of dry matter during the dough stage (i.e. 24–30 DAP), with both Zn and dry matter accumulating at relatively constant proportions from the dent stage to kernel maturity (Abendroth et al., 2011).

There was a substantially higher Zn content in ‘Hybrix 5’ sweetcorn kernels compared with that in ‘Thai Floury 2’ maize kernels at 18 DAP (44 % higher), but this progressively decreased to parity by 24 DAP (5.2 µg of Zn) and to 16 % lower kernel Zn content by 56 DAP (Table 2). The rate of Zn accumulation in ‘Thai Floury 2’ kernels (0.49 µg Zn d^–1) was 53 % higher than that recorded for ‘Hybrix 5’ (0.32 µg Zn d^–1) over the period from 18 to 28 DAP and, while the rates slowed in both varieties from 28 to 56 DAP, ‘Thai Floury 2’ (0.14 µg Zn d^–1) was still able to maintain a 27 % higher rate of Zn accumulation than ‘Hybrix 5’ (0.11 µg Zn d^–1).

While embryo and endosperm tissues were not analysed separately at 56 DAP (Table 2), there were also differences in the relative contribution of the embryo and the endosperm Zn from the whole kernel Zn content between the two varieties. While the proportion of Zn found in the embryo tissue increased continuously as the kernels matured in both varieties (Table 2), the embryos of ‘Thai Floury 2’ (28–49 % of kernel Zn) always represented a substantially higher proportion of kernel Zn than those of ‘Hybrix 5’ (18–33 % of kernel Zn).

Analyses of standards and tissue samples using XANES

The speciation of Zn within the embryos, endosperms and whole kernels of the ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize varieties was examined using synchrotron-based XAS and compared with the spectra obtained from nine standard compounds. First, we examined the spectra from the nine standard compounds. These spectra were visually different, with differences in both the energy and height of white line peaks, as well as differences in the spectral shapes and features (Fig. 1). For example, the energy values corresponding to the white line peaks ranged from 9663.6 eV (Zn-cysteine) to 9668.9 eV (metallic Zn), while the white line peaks of Zn-phytate and Zn-citrate (and to a lesser extent Zn-cysteine) were very pronounced compared with those of the other standards.

Fig. 1.

Average of the normalized Zn K-edge XANES spectra for the nine standard compounds analysed at pH 7. The vertical lines are at 9664.8 eV and 9666.8 eV, which correspond to the white line peaks of Zn-phytate and Zn-histidine, respectively.

The spectra obtained from samples of embryos, endosperms and whole kernels of ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize at 21, 28 and 56 DAP were then examined (Fig. 2). Noticeable differences were observed between the spectra for the embryos and the endosperms. All four embryo spectra were almost identical, with a distinct white line peak observed at 9664.5 eV, being quite similar to that obtained for the Zn-phytate standard (9664.8 eV). In contrast, the spectra from the endosperm samples had less pronounced white line peaks at 9666.8 eV (Fig. 2), which was most similar to that obtained from the Zn-histidine standard which shared the same white line peak energy value (i.e. 9666.8 eV) and also a gradual slope and broader peak.

Fig. 2.

Average normalized Zn K-edge XANES spectra for ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize (Zea mays) embryo, endosperm and whole kernel at 21, 28 and 56 days after pollination. Spectra of sweetcorn and maize are in red and green, respectively. Data are also presented for relevant standard compounds. The vertical lines are at 9664.8 eV and 9666.8 eV, which correspond to the white line peaks of Zn-phytate and Zn-histidine, respectively.

The LCF analysis predicted that 88–92 % of the Zn within the embryo was present as Zn-phytate, with the remaining 8–12 % of the Zn present as Zn-cysteine (R-factor = 0.00056–0.00062). Meanwhile, the LCF analysis predicted that 82–87 % of the Zn within the sweetcorn endosperm was Zn-histidine, with the remaining 13–18 % Zn-phytate (Table 3; Supplementary data Fig. S1).

Table 3.

Linear combination fitting predictions of Zn speciation composition in embryos, endosperms and whole kernels of ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize (Zea mays) at 21, 28 and 56 days after pollination (DAP)

Variety	‘Hybrix 5’ sweetcorn						‘Thai Floury 2’ maize
Tissue	Embryo		Endosperm		Whole kernel		Embryo		Endosperm		Whole kernel
DAP	21	28	21	28	21	28	21	28	21	28	21	28
Speciation (%)
Zn-phytate	88	88	13	18	30	41	88	92	–	83	27	87
Zn-cysteine	12	12	–	–	3	4	12	8	8	15	9	12
Zn-histidine	–	–	87	82	67	55	–	–	92	2	63	1
R-factor	0.00056	0.00064	0.0014	0.00099	–	–	0.00062	0.00065	0.00086	0.0061	–	–

There are no R-factor values for whole kernel samples, as the speciation estimates are calculated values from individual tissue analysis instead of LCF predictions.

Interestingly, there was a shift in Zn speciation in the maize endosperm between 21 and 28 DAP from a Zn-histidine-dominant composition to a Zn-phytate-dominant composition (Fig. 2B). Indeed, the LCF analysis also predicted 92 % Zn-histidine and 8 % Zn-phytate at 21 DAP, which then changed to 2 % Zn-histidine and 83 % Zn-phytate at 28 DAP, with the remaining 15 % predicted to be Zn-cysteine (Table 3). However, it is emphasized that although the LCF prediction for maize endosperm at 21 DAP was relatively good (R-factor = 0.00086), the R-factor for the 28 DAP maize endosperm prediction was approximately seven times poorer (R-factor = 0.0061; Table 3), indicating that the fit was not ideal. Therefore, though the shift in 28 DAP maize endosperm to contain a larger proportion of Zn-phytate is certain, the exact speciation is uncertain and may include other forms of Zn not analysed as a reference standard.

Quantitative estimation of Zn speciation in homogenized whole kernel samples was challenging, due to the marked contrast in tissue Zn concentration and Zn speciation between the kernel constituents (i.e. embryo and endosperm). This resulted in relatively poor LCF predictions (R-factor = 0.0022–0.0037) and low confidence in predictions about Zn speciation from such samples. As a result, Zn speciation in whole kernels (Table 3) was calculated on the basis of the proportional dry matter contribution of embryo and endosperm.

Analyses of Zn, P and S distribution in sweetcorn kernels using XFM

Tri-colour images (red–green–blue, RGB) were used to further examine the association between Zn, P and S (Fig. 3). In all immature and mature (Fig. 3H) samples, the highest concentrations of Zn, P and S were found to accumulate within the embryo. Although in immature kernels the Zn accumulated predominantly in the scutellum of the embryo (Fig. 3E–G), in mature kernels it also accumulated in the shoot apical meristem (Fig. 3H). While P and S were found in both the scutellum and embryo axis at all maturity stages, P mainly accumulated in the scutellum whereas S mainly accumulated in the embryo axis.

Fig. 3.

Light micrographs and RGB co-localization maps of Zn (red), P (green) and S (blue) in ‘Hybrix 5’ sweetcorn (Zea mays) whole kernels from the front view (A and E), side view (B and F), the embryo (C and G) at 21 DAP, and whole kernel front view at 56 DAP (D and H). 1, aleurone layer; 2, endosperm; 3, scutellum; 4, embryo axis; 5, basal plate; 6, shoot apical meristem; 7, black abscission zone. Micrographs were taken using a Leica MZ6 dissection microscope, objective achromat ×1.0, working distance = 90 mm.

Interestingly although elevated concentrations of Zn and S were found outside of the embryo, only low concentrations of P were detected in the other tissue constituents at any maturity stage. However, there seems to be little co-localization between the Zn and S in these tissues, with Zn accumulating in the aleurone layer and S accumulating in the endosperm. This was visible at the immature kernel stage (Fig. 3E) and exceptionally distinguishable at the mature kernel stage (Fig. 3H). These observations support the finding that Zn is complexed with phytate and cysteine (S-based ligand) in the embryo, whereas it is unlikely to be complexed with cysteine in the endosperm but rather with another compound such as histidine (N-based ligand).

Discussion

Bioavailability of Zn in the embryo and endosperm

This study provided in situ analyses of Zn speciation in sweetcorn and maize kernels using XAS, thereby minimizing possible experimental artefacts induced by sample preparation processes such as chemical digestion in other techniques. This study revealed that Zn obtained from the consumption of endosperms is likely to be more bioavailable compared with Zn from embryos. This is because the Zn in the endosperms of immature (21 DAP) sweetcorn and maize kernels was largely associated with N-based ligands such as histidine, thereby suggesting a higher bioavailability to consumers (Snedeker and Greger, 1983; Solomons, 2001; La Frano et al., 2014), whereas in the embryo the Zn was present as Zn-phytate (Fig. 2). This expands the findings of a previous XAS study which only examined sweetcorn at a single level of maturity in a market survey (Cheah et al., 2019a) to include maize varieties. The high proportion of Zn-phytate within the embryo is also consistent with previous studies reporting that most of the maize phytate content is found in the embryo (O’Dell, 1972).

The observation that the Zn in the endosperm is complexed with an N-based ligand is consistent with previous studies reporting co-localization of Zn with proteins in various cereal grains (Kutman et al., 2010; Lombi et al., 2011; Kyriacou et al., 2014). Indeed, there have been multiple studies demonstrating the association between Zn and protein in wheat grains (Peck et al., 2008; Peleg et al., 2008). Furthermore, other studies have shown that there is a common genetic basis controlling the concentration of Zn and protein in grains (Uauy et al., 2006; Distelfeld et al., 2007; Xu et al., 2012), and, when certain genes are downregulated, remobilization of Zn and N from vegetative tissues into the grain was impaired (Waters et al., 2009). In maize, some studies have also shown that the opaque-2 gene concurrently improves kernel content of Zn and zein proteins such as lysine and tryptophan (Arnold et al., 1977; Welch et al., 1993). Unfortunately, those studies were mainly focused on improving protein quality in maize; hence; no further studies were conducted to pursue Zn biofortification in those varieties. Nonetheless, these results confirmed that N-containing ligands play an important role in complexing with Zn in plants (Broadley et al., 2007). It is also worth noting that the presence of Zn bound with cysteine in the 28 DAP maize endosperm is consistent with previous findings that Zn is complexed with S-based ligands in various cereal grains (Persson et al., 2009, 2016; Hansen et al., 2012).

Changes in endosperm Zn speciation as maize kernels mature

Importantly, the Zn speciation of maize endosperm changed as the kernel matured, shifting from approx. 90 % Zn-histidine at 21 DAP to approx 83 % Zn-phytate at 28 DAP, significantly reducing the bioavailability of the Zn in maize endosperm (Table 3). This increased complexation between Zn and phytate ligands could have possibly occurred in the aleurone layer (which was not separated from the endosperm tissue during analysis in this study), as P was also found to accumulate in the aleurone tissue in mature maize kernels (Cheah et al., 2019b). This shift in endosperm Zn speciation is physiologically feasible, as it was reported that phytate-P concentration in maize kernels rapidly increase from 0.1 to 1.6 mg kg^–1 d. wt between 15 and 30 DAP, which then continued to increase to 2.1 mg kg^–1 at 40 DAP and to 2.6 mg kg^–1 at full maturity (Raboy et al., 2000). It is therefore possible that a higher percentage of endosperm Zn in a mature kernel (56 DAP) from this study will eventually be in the form of Zn-phytate, which could potentially explain why despite having a considerable amount of Zn stored within the endosperm (Table 2), bioavailability of Zn in maize is low (O’Dell, 1972; Solomons, 2001; Maqbool and Beshir, 2019). This has major implications for Zn biofortification in maize which is often consumed at the mature kernel stage, as it suggests that minimal dietary benefit can be derived from increasing Zn concentration in maize kernels.

Interestingly, in the sweetcorn endosperm, the increase in Zn-phytate composition between 21 and 28 DAP was minimal (Fig. 2B; Table 3). Findings from the XFM analysis suggest that this pattern persists up to kernel maturity at 56 DAP, as minimal P was detected in the endosperm or aleurone layer where Zn accumulated (Fig. 3). This may provide genetic clues to maintaining the bioavailability of Zn in the endosperm, which could be incorporated into maize germplasm to improve the nutritional value of this staple food.

Benefits of Zn biofortification of sweetcorn and maize

It was initially hypothesized that the advantage of using sweetcorn as a target crop for Zn biofortification was that the entire kernel is consumed, whereas in maize the embryo and the aleurone layers are removed during processing (Reddy and Love, 1999; Gwirtz and Garcia-Casal, 2014; Suri and Tanumihardjo, 2016). This is important as Zn accumulates at high concentration in the embryo and aleurone layer tissues (Cheah et al., 2019b). However, this study revealed that the true benefit of obtaining Zn from a sweetcorn diet is the consumption of immature kernels, as compared with the consumption of mature kernels for maize. In immature kernels, an increase in Zn content results in 72–77 % being stored in the endosperm and the remaining 23–28 % being stored in the embryo (Table 2). Since Zn in the endosperm of immature kernels appears to be bioavailable due to complexation with N-based ligand such as histidine (Fig. 2b), increasing the Zn concentration in sweetcorn kernels should result in improved Zn nutrition for consumers.

Conversely, biofortification of Zn in maize for human consumption faces a few challenges. First, since the proportion of Zn contributed by the embryo increases as the kernel matures, our data suggest that potentially at least 49 % of the Zn content in mature maize kernels will be removed with those embryos during food processing (Table 2). This is consistent with earlier studies that reported that removal of the embryo during food processing significantly reduced whole kernel Zn content by 80–85 % (Pedersen and Eggum, 1983; Bressani et al., 2002; Oghbaei and Prakash, 2016). Secondly, the shift in maize endosperm Zn speciation from complexation with an N-based ligand to Zn-phytate as the kernel matures (Fig. 2B) is likely to negate the benefits of increasing Zn concentration in maize kernels, as dietary Zn obtained from a mature maize kernel will have low bioavailability.

It is interesting to note that the highest rate of kernel Zn accumulation in both sweetcorn and maize varieties occurred during the 21–24 DAP period (Table 2). The highest Zn concentration for both maize embryo and endosperm tissues (and therefore also whole kernel Zn concentration) was also observed at 24 DAP, which coincides with the end of the milk stage of kernel development (Abendroth et al., 2011). Similar observations have been made in wheat grain whereby the highest Zn concentration was also observed at the milk stage, although in wheat it was at the early milk stage (Ozturk et al., 2006).

Conclusions

This study determined that >88 % of Zn accumulated in the embryo of sweetcorn and maize kernels at any maturity stage is as Zn-phytate. Meanwhile, in the endosperm of immature kernels (21 DAP), >87 % of the Zn is complexed with an N-based ligand such as Zn-histidine. Although these forms and distributions largely remain present as sweetcorn kernels mature, speciation in maize endosperm changes such that a much larger proportion of Zn occurs as Zn-phytate by 28 DAP. This is likely to substantially reduce the bioavailability of Zn supplied in a maize diet, undermining the effectiveness of Zn biofortification in maize kernels. Conversely, 72–77 % of the Zn accumulated in sweetcorn kernels at consumption maturity (i.e. 21–24 DAP) is stored in the endosperm and therefore is likely to be bioavailable for consumers. This highlights the potential of sweetcorn as a biofortification target to enhance human dietary Zn intake.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of Figure S1: average normalized Zn K-edge XANES spectra for whole kernel, embryo and endosperm of ‘Hybrix 5’ sweetcorn and ‘Thai Floury 2’ maize at 21, 28 and 56 days after pollination.

FUNDING

MRCAT operations are supported by the US Department of Energy (DOE) and the MRCAT member institutions. This research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Although the Environmental Protection Agency (EPA) contributed to this article, the research presented was not performed by or funded by EPA and was not subject to EPA’s quality system requirements. Consequently, the views, interpretations and conclusions expressed in this article are solely those of the authors and do not necessarily reflect or represent EPA’s views or policies. Part of this research was also undertaken on the X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron, part of ANSTO. This work was supported by the Australian Government Research Training Program (RTP) Scholarship.