Carotenoid and Tocochromanol Profiles during Kernel Development

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Agricultural and Environmental Chemistry

Carotenoid and tocochromanol profiles during kernel development make consumption of biofortified “fresh” maize an option to improve micronutrient nutrition Luisa Cabrera Soto, Kevin Vail Pixley, Aldo Rosales-Nolasco, Luis Alberto Galicia-Flores, and Natalia Palacios-Rojas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01886 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Journal of Agricultural and Food Chemistry

Carotenoid and tocochromanol profiles during kernel development make consumption of biofortified “fresh” maize an option to improve micronutrient nutrition Luisa Cabrera-Sotoa, Kevin V. Pixleya, Aldo Rosales-Nolascoa, Luis A. Galicia-Floresa, Natalia Palacios-Rojasa* a

International Maize and Wheat Improvement Center (CIMMYT), CIMMYT Research Station, Km. 45

Carretera Mexico-Veracruz, El Batán, Texcoco, CP 56237, Edo. de México, México.

*Tel.: +52 595 95 21900; E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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Biofortification is a strategy to contribute reducing micronutrient malnutrition. The aim of this study

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was to investigate whether consumption of biofortified fresh maize can supply nutritionally meaningful

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amounts of provitamin A carotenoids (PVA), zinc, lysine and tryptophan. The accumulation patterns for

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PVA and tocochromanols compounds in developing grain of 23 PVA hybrids was studied and it was

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nutritionally meaningful amounts of those compounds were found in grain by milk stage, when fresh

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maize is eaten. The highest PVA and tocochromanols accumulation occurred by physiological maturity.

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The percent apparent retention in boiled fresh maize was 92, 117, 99 and 66% for PVA, zinc, lysine and

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tryptophan, respectively. Consumption of 0.5 to 2 ears of fresh maize daily could supply 33-62.2%, 11-

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24% and more than 85% of the estimated average requirement of PVA, tryptophan and zinc,

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respectively. The results indicate that eating biofortified fresh maize can contribute to improved

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micronutrient nutrition.

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KEYWORDS: biofortification, quality protein maize, provitamin A, tocochromanols, kernel zinc,

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vitamin A, vitamin E.

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INTRODUCTION

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Maize contributes over 20 percent of total calories in human diets in 21 low-income countries, and

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over 30 percent in 12 countries with more than 310 million people, mainly in Mesoamerica, Sub-

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Saharan Africa and Southeast Asia1.Unfortunately, the amounts of some essential nutrients present in

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maize kernels are inadequate for consumers that rely on maize as a major food source2. For example,

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from a human nutrition context, maize kernels are deficient in provitamin A (PVA), zinc, vitamin E

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(tocochromanols), iron, iodine, and the essential amino acids lysine, methionine and tryptophan. White

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maize is the most common maize consumed by humans in Africa and Mesoamerica, but it is devoid of

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carotenoids (including PVA carotenoids).

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Biofortification, the process of breeding nutrients into staple food crops, provides a sustainable

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strategy for delivering micronutrients and improving the nutritional status of malnourished populations

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in developing countries3,4,5,6. In the case of maize, quality protein maize (QPM) and PVA maize have

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been developed, availed, and their consumption promoted in different countries aiming to contribute to

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alleviating essential amino acid and vitamin A deficiencies, respectively7,8,9. Breeding efforts to develop

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high zinc maize are also currently underway7,10.

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When view of its nutritional value as food or feed, it is important to consider that the nutrient

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composition of maize varies during kernel development. Maize grain filling comprises seven stages that

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vary in timing and duration depending on agro-ecological conditions and genetic characteristics of the

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maize. In general, the stages are silking (SS); kernel blister (KB) (12-18 days after pollination (DAP));

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milk stage (MS) (19-26 DAP); dough stage (DS) (28-35 DAP); kernel dent stage (KDS) (35-46 DAP);

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physiological maturity (PM) (61-65 DAP); and harvest maturity (HM), when harvest can occur with

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minimal kernel damage, or around 25% kernel moisture content (often more than 75 DAP)11.



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“Green” maize or hereafter “fresh” maize, refers to ears or kernels from any variety of maize that are

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harvested at or soon after the milky stage and usually are cooked by roasting or boiling2. Fresh maize is

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potentially a high value product, fetching up to 5 times the price of grain. While China, Brazil, India,

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Mexico, Nigeria, Tanzania and Kenya are the top fresh maize producing and consuming countries12, it

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also is an important food in many other sub-Saharan countries. The appearance of fresh maize often

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marks the end of the “hungry season”, when previous harvest has been consumed and the current crop is

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still in the field13. Despite its importance, little is known about the nutritional value of fresh maize.

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Reports for macronutrient and mineral retention for roasted and boiled fresh maize have varied for

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yellow, white and PVA varieties13,14,15.

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The accumulation of nutritionally important compounds like carotenoids has been monitored in

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temperate maize during the first 28 DAP16,17, which corresponds to the period from early maturation of

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the endosperm until kernel dough stage. Ortiz et al.18 reported the accumulation of carotenoids during

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the late stages of grain filling, kernel dent, physiological maturity and harvest maturity. However, there

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are no published reports on the accumulation of carotenoids and tocochromanols (vitamin E) throughout

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all grain filling stages for PVA biofortified maize. This knowledge will help determine the potential

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usefulness of PVA fresh maize as a source of nutrients for consumers, and will also contribute to further

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understanding maize endosperm development.

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The objectives of this study were to: 1) evaluate carotenoid and tocochromanol accumulation

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throughout grain filling stages of PVA maize, and 2) estimate the retention of PVA carotenoids, zinc,

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lysine and tryptophan following boiling of fresh maize of PVA, high Zn and QPM varieties,

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respectively.

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MATERIALS AND METHODS

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Plant materials

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Two experiments were conducted using either PVA, high-Zn or QPM hybrids from subtropical and

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tropical breeding programs of the International Maize and Wheat Improvement Center (CIMMYT). For

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experiment 1, twenty-three subtropical PVA hybrids (PVAH) with very similar flowering dates and two

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commercial yellow hybrids (CYH) were grown in 2 replicates at Mexico’s National Institute of Forestry,

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Agriculture and Livestock Research’s (INIFAP) experiment station at Celaya, Guanajuato (CE)

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(20°26’N, 103°19’W, 1750 m above sea level, average annual temperature 19 °C, average annual

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precipitation 700 mm) during summer 2012.

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Grain sampling dates were scheduled to correspond with Nielsen et al.’s grain filling stages for

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temperate dent maize. The counting of “days after pollination” (DAP) started from the date when 50%

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of the plants were flowering (silk started to emerge, and pollen present). To account for thermal

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differences during grain filling, we estimated the growing degree-days (GDD) for the given DAP of

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each stage. A mean daily GDD was calculated as follows:

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GDD= [0.5*(Tmax+Tmin)]-Tbase

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where Tmax was the daily high, Tmin was the daily low, and Tbase was 10 °C, as previously described and

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used for maize19 . Temperature data were obtained from the meteorological station at the experimental

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site (CE).

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Grain samples were collected from controlled self-pollinated ears based on number of DAP.

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Flowering time of the genotypes was similar and pollination dates were between 0-4 days apart. Samples

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consisted of one ear per replicate. GDD for each sampling was the average of the GDD for the sampling



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date. Ear samples were collected at: pre-blister (8-12 DAP, 1132 GDD); blister (12-16 DAP, 1165 GDD,

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80-85% kernel moisture); kernel milk (16-20 DAP, 1199 GDD, 75-80% kernel moisture); kernel dough

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(25-29 DAP, 1277 GDD, 60-65% kernel moisture); kernel dent (36-40 DAP, 1381 GDD, 50-55% kernel

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moisture), physiological maturity (58-62 DAP, 1567 GDD, 30-35% kernel moisture) and harvest

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maturity (63-67 DAP, 1596 GDD, 18-25% kernel moisture).

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For experiment 2, ten QPM hybrids (QPM), ten high-Zn hybrids (high-Zn) and ten PVAH were

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grown in 2 replicates at CIMMYT’s research station near Agua Fria, Puebla, Mexico (20°32´N, 97°28´

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W, 110 m above sea level, average annual temperature 22 °C, average annual precipitation 1200 mm)

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during winter cycle of 2013. Three to five ears per replicate per genotype were collected between kernel

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milk and dough stageand used for kernel sampling and fresh maize cooking. In addition, three to five

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ears per replicate per genotype were collected at harvest maturity (>63 DAP, 18-25% kernel moisture).

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Kernels from the middle part of the ears were sampled in duplicate for micronutrient analysis. All

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samples with the exception of kernels from harvest maturity stage were freeze-dried on liquid nitrogen

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before transporting them to the laboratory. Prior to the chemical analysis, samples were lyophilized in

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darkness for 6 days at -80 °C using a VirTis Benchtop 2KBTXL freeze-dryer. All grain samples were

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stored at -80 °C until the day of analysis. Ears for cooking were harvested and transported on ice (6-8

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°C) to the laboratory prior to cooking.

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Cooking of fresh maize

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In Mexico and other parts of Latin America, it is common to keep the inner husk leaves attached to

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the ear when boiling fresh maize. Three maize ears per hybrid were boiled separately following this

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practice. Samples were placed and covered with deionized water in a traditional stock pot, heated to


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boiling, and cooked for 35-40 minutes. After cooling, the cooked grains were cut from each cob and

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stored in falcon tubes at -80 °C. Samples were lyophilized as described above.

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Determination of carotenoids and tocochromanols

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Carotenoids (lutein (LUT), zeaxanthin (ZEA), β-cryptoxanthin (BCX) and β-carotene (BC)) and

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tocochromanols (α-tocopherol (ATP), γ-tocopherol (GTP) and α-tocotrienol (AT3)) were analyzed for

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whole kernel samples of each grain filling stage. Extraction and ultra-performance liquid

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chromatography (UPLC) were performed as described by Muzhingi et al. (2016). LUT, ZEA, BCX, BC

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and total PVA concentrations (PVA was computed as: all-trans-BC + 0.5(13-cis-BC + 9-cis-BC +

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BCX)) were measured and reported in µg/g of kernel dry weight (DW) (Muzhingi et al., 2016).

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Lysine and tryptophan analysis in maize kernels Lysine and tryptophan content in maize grain were determined as described in Galicia et al. and Nurit et al., respectively. Lysine and tryptophan content were expressed as percent of kernel dry matter.

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Zinc concentration in maize kernels

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Zinc concentration was determined by wet digestion as described by Galicia et al.20 with minor

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modifications. Briefly, samples (300 mg) were weighed into 100 mL Pyrex tubes. Digestion was

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initiated by adding 5 mL HNO3/HClO4 mixture (9:1 v/v). Samples were vortexed to thoroughly wet the

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material, covered with polyethylene wrap and then allowed to pre-digest overnight at room temperature

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under a fume hood. Digestion was performed by gradually heating the digestion block from 80 °C to

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225 °C during 4 h. After cooling, 10 mL of HNO3 were added and the sample was mixed prior to

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analysis by ICP-OES.



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Percent apparent retention (%AR) for PVA, lysine, tryptophan and zinc was calculated as21 :

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%AR =

Nutrient content per g of cooked food (dry basis) × 100 Nutrient content per g of raw food (dry basis)

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Estimated Average Requirement of vitamin A, Zinc and tryptophan supplied by fresh maize

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We calculated the percentage of the USDA’s estimated average requirement (EAR)22 of vitamin A

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that could be supplied by boiled fresh maize, based on the retinol activity equivalents (RAE) and the

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PVA retention values determined as described above. EAR for zinc and tryptophan were also calculated

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based on the retention of those compounds in boiled fresh maize. Tryptophan values were estimated

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based on niacin equivalents23.

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Statistical analysis Analysis of variance (ANOVA) and Tukey’s test (P < 0.05) to identify significant differences among groups were conducted using SAS version 9.

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RESULTS AND DISCUSSION

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Carotenoid accumulation during kernel development

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The chronological trend in carotenoid accumulation was similar for all genotypes (Fig. 1a) despite

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genotypic differences in total and individual carotenoid contents (Suppl. Table 1). Total carotenoid

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concentration increased significantly up to PM and decreased slightly or remained unchanged until HM

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(Suppl. Table 1; Fig 1a). Our data agreed with published results that LUT and ZEA are the major

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carotenoids in maize8. BCX and BC content were higher in the PVA biofortified hybrids than in the

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CYH, and were within the ranges previously reported24,25. LUT and ZEA content increased until the 8 ACS Paragon Plus Environment

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KDS, after which they decreased by HM stage (Fig. 1a). BCX, all-trans-BC and 13-cis-BC also

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increased throughout the grain filling stages, reached their highest contents at the PM stage, and

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decreased significantly by HM. Only 9-cis-BC reached its maximum concentration and remained

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unchanged after the kernel dent stage (Fig. 1a).

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The percentage of PVA carotenoids relative to total carotenoid content increased significantly up to

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PM stage (Fig. 1b), and then slightly but significantly decreased by HM, as occurred also for total

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carotenoids. Such decreases did not occur for all genotypes, and all-trans-BC was the PVA carotenoid

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that decreased the most (Suppl. Table 1). In most of the genotypes, including the CYH, total carotenoid

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accumulation reached its peak at DS-KDS stages, while PVA carotenoid content was maximum at PM

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(Fig. 1b; Suppl. Table 1). Ortiz et al. found no significant changes in PVA carotenoids concentrations

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for kernels of temperate/mid-altitude PVA maize collected 45, 52 and 85 DAP, which may have

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corresponded to KDS, PM and HM, respectively. This difference, i.e. that we observed a peak of PVA

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carotenoids concentrations between KDS and HM whereas Ortiz et al.18 observed no changes in PVA

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carotenoids during this time, may have been due to small differences in kernel maturity stages when

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samples were harvested, differences among germplasm used, or environmental effects (high latitude

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(long day length) as compared to our study). Unfortunately, no GDD or detailed kernel stage definitions

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were provided in the Ortiz et al.18 report.

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A trend of carotenoid accumulation throughout the grain filling stages is consistent with reports of

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gene expression during endosperm development. Genes controlling steps upstream of the carotenoid

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pathway (DXS3, DXR, HDR, GGPPS1, PSY; Suppl. Fig. 1) are positively correlated with carotenoid

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content, while ZEP1 and ZEP2 expression are inversely correlated with carotenoid content in kernels 25

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DAP (KDS) 16, 17.



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A pronounced decrease, typically 40 to 60 percent, in PVA carotenoid concentrations soon after

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harvesting has been reported, as well as the existence of genotypic differences in the magnitude of such

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decrease18,26,27. Our data indicate that the decline in PVA starts before kernels reach harvest maturity and

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confirms both that the magnitude of decrease depends on the genotype and that BC decays more than

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BCX26,27 (Suppl. Table 1).

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Apocarotenoids are the oxidation products of carotenoids, and in plants these reactions are catalyzed Non-enzymatic processes also degrade carotenoids28. Both

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by carotenoid dioxygenases (CCDs).

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mechanisms are important determinants of carotenoid concentrations in grain and food. Degradation of

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carotenoids can occur at any stage of kernel development; however, given the high water activity (>0.8)

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at PM and HM, carotenoid catabolism enzymes may be particularly active at these stages28,29.

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Endosperm development in grasses has three main phases, known as early development, differentiation

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and maturation; they can overlap considerably and they normally occur within the silking and grain

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filling stages30. Gonzalez-Jorge et al.31 have shown that catabolism mediated by the carotenoid cleavage

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enzymes (CCD1, CCD4, CCD7 and CCD8) and the 9-cis-epoxycarotenoid dioxygenases (NCED2,

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NCED3, NCED5, NCED6 and NCED9) affects carotenoid composition and content in Arabidopsis

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seeds. Higher mRNA levels of ccd4, ccd7 and nced9 at 25 DAP compared to 20 DAP suggests that

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carotenoid catabolism starts early during endosperm development31. Genotypic variation for the effects

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of genes encoding such enzymes on carotenoid concentrations has been suggested in maize24, 32.

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Natural variation in ZEP gene expression during seed development affects carotenoid composition,

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stability, and ultimately carotenoid content in seeds17,33. Arabidopsis mutants lacking ZEP activity

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showed a remarkable increase in seed carotenoid contents compared to the wild type, mainly for ZEA

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but also LUT and BC33. Additionally, in maize, by 25 DAP approximately the top half of the endosperm

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is dead and programmed cell death (PCD), which is known to be affected by abscisic acid (a carotenoid


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derived hormone), continues up to 40-44 DAP (PM stage)34,35. Thus, as kernel development approaches

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physiological maturity, transcription of enzymes involved in catabolism is ongoing while carotenoid

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biosynthesis may have stopped, and both carotenoid catabolism and non-enzymatic degradation

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mechanisms can occur. Although endosperm development has been broadly characterized for maize,

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there is limited information on the cellular and molecular processes that are very important for the

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improvement of grain quality and cereal derived food products30. Recently, Schaub et al, have

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investigated BC degradation pathways over time in golden rice and orange flesh sweet potato,

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demonstrating a major role of non-enzymatic mechanisms and confirming the quantitative importance of

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highly oxidized BC polymers, reported previously in dried plant tissues28. PVA maize germplasm can be

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a valuable resource for understanding the carotenoid pathway and elucidating strategies to minimize

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carotenoid decay. More detailed transcript studies, such as analyses of downstream carotenoid

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biosynthetic compounds like violaxanthin, anterazanthin, the products of ZEP-mediated epoxidation of

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zeaxanthin, and evaluation of apocarotenoids, especially after PM, will provide additional useful

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insights.

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Our results clearly indicate that researchers developing PVA maize must measure carotenoid content

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of materials either at physiological maturity or consistently at the same time after harvest maturity to

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enable valid comparisons among materials. Within the PVA breeding program at CIMMYT, we analyze

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samples 4 to 6 weeks after HM, when a carotenoid content has stabilized26. It is recommended to store

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the ears or grain at room temperature (20-25 °C), low ambient moisture (

Carotenoid and Tocochromanol Profiles during Kernel Development

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