Wheat Biofortification: Progress and Challenges

Ruchi Bansal*, Jyoti Kumari, Rakesh Bhardwaj, Sundeep Kumar

National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India 110012

*Corresponding author: Ruchi Bansal ( E-mail: ruchibansal06@gmail.com)

Hidden hunger is a substantial public health problem. One of three people in the world suffers from hidden hunger caused by a lack of micronutrients in their diets. Deficiency of micronutrients leads to stunted growth in children, reduction in immunity and work efficiency in adults, in particular women. Iron and zinc have been recognized as the most important among micronutrients. The World Health Organization estimated that approximately 25% (one fourth) of the world’s population suffers from anemia (WHO, 2008), while, Zn-deficiency lead to estimated annual deaths of 433,000 children under the age of five (WHO, 2009). Iron deficiency causes anemia and leads to impaired mental development in children, reduced capacity for physical labour in adults. Iron-deficiency anemia was so severe that it led to the loss of over 46,000 disability adjusted life years in 2010 alone (Murray and Lopez, 2013). Zinc is crucial for immunity, control of diabetes, healing, digestion, reproduction and physical growth. An estimated 17.3% of world’s population is at risk of zinc deficiency (Wessells and Brown, 2012). Now the fortification programs are drawing attention of policymakers and were ranked third among the most effective ways to address the developmental changes in Copenhagen consensus 2008 (Gomez-Galera et al., 2010). Different strategies to solve the problem are supplementation, dietary diversification, fortification, agronomic fortification. However, these strategies require continuous investment and infrastructure. Biofortification provides a viable option to combat malnutrition in a cost effective manner.

What is biofortification?

Biofortification is a process for increasing the bioavailable concentrations of essential elements in the edible portions of crops through agronomic intervention or genetic selection (White and Broadley 2005). Most of the people are dependent on cereals and legumes as their primary dietary source of these micronutrients, therefore, biofortification of staple crops is an effective strategy, which ensures the consumption of essential micronutrients in a cost effective manner. Wheat is the major staple food crop of the world, which contributes up to 28% of the world edible dry matter production and up to 60% of daily energy intake in several developing countries (Wang et al., 2011). Therefore, the composition and nutritional quality of the wheat grain have a significant impact on human health (Chatzav et al., 2010; Wang et al., 2011).

Iron and zinc uptake and translocation to the grain

The accumulation of micronutrients in cereal grains is a complex process, which depends on the availability of the micronutrient in the soil, its mobilization from the soil, absorption by roots, translocation, remobilization from vegetative tissues, and deposition in bioavailable forms in cereal grains. The nutrient uptake and utilization efficiency depends on the genotype as well as the environment. The whole process is controlled by numerous genes (Bouis and Welch 2010).

Fe and Zn uptake pathways have been studied in details in rice, maize and barley. Fe and Zn uptake from the rhizosphere occurs by two processes. One is the direct uptake of Fe2+ and Zn2+ by ZRT, IRT-like proteins referred as ZIPs. Another is by secretion of phytosiderophores, which chelate Fe cations and these chelated forms are taken up by yellow stripe like (YSL) transporters (Sperotto et al., 2012). In wheat, Fe is taken up in the chelated form. The whole process involves the transporters of multi-gene families. Metal chelators are important for radial movement of Fe and Zn through the root (Rellán-Álvarez et al., 2010; Deinlein et al., 2012). In xylem, Zn can move either as a cation or in a complex with organic acids such as citrate (Lu et al., 2013), while, Fe is chelated by citrate (Rellán-Álvarez et al., 2010). ZIP/YSL family proteins facilitate the transfer from xylem to phloem in the basal part of the shoot or during remobilization from the leaves during grain filling. In wheat all nutrients enter the grain through the phloem because the xylem is dis-continuous. In the phloem, Fe and Zn are transported as complexes with nicotianamine or small proteins. Transporters belonging to  ZIP, YSL, and metal tolerance protein (MTP) families have been suggested to be involved in the transport of these cations from the maternal tissue into the endosperm cavity, aleurone layer and embryo (Borg et al., 2012; Tauris et al., 2009). In wheat grain, most of the Fe and Zn are mainly deposited in the aleurone layer, which is lost during the milling process. Furthermore, Fe in these tissues is deposited mainly in protein storage vacuoles, where it is bound to phytate. In the bound state, it is poorly bioavailable (Borg et al., 2012). Thus, the bioavailbility of these micronutrients not only depends on their total content in the grain, but also on their localization.

Micronutrients bioavailability

Bioavailability of the micronutrients depends upon the interaction between factors depending on the grain composition. The percent bioavailability of Fe and Zn in staple food crop seeds and grains is as low as 5% and 25%, respectively (Bouis and Welch 2010). Thus, bioavailability of micronutrients is an important factor despite the total concentration in the grain. The grains of cereal crops contain various factors, such as phytic acid and tannins (Guttieri et al. 2006), which can reduce the bioavailability of micronutrients. Phytic acid forms a complex with the mineral elements and reduces their bioavailability leading to Fe and Zn deficiency (Liu et al. 2006).  Some factors, which stimulate the absorption of micronutrients are, vitamin C, pro-vitamin A, hemoglobin and various organic and amino acids.

The composition of the grain with respect to micronutrients and all other factors depends on the genetic make up and environmental conditions (Welch and Graham 2004; White and Broadley 2005). Zn fertigation affects the phytic acid concentration in the wheat grain. Phytic acid concentration ranged from 7 to 12 mg g-1 with Zn fertilization and 8 to 13 mg g-1 without Zn fertilization (Erdal et al. 2002). Significant genotypic variation with respect to phytic acid content was also recorded in wheat grain (Raboy et al. 1991; Erdal et al. 2002; Welch and Graham 2004; White and Broadley 2005).

Genetic variation for micronutrients

Genetic variation in the trait of interest is the basis for crop improvement through plant breeding strategies. There are significant differences in Zn efficiency (the ratio of yield under Zn deficiency versus Zn fertilization) between wheat and its relatives, and individual chromosomes from genera Secale, Agropyron, and Haynaldia carry major genes that control Zn efficiency. It was reported that the Zn efficiency of cereals declines in the following order: rye > triticale > barley > bread wheat > oat > durum wheat (Schlegel et al. 1998).

Cakmak et al. 1999 reported that Zn efficiency varied with the evolution of wheat and for diploid (AA), tetraploid (AABB), and hexaploid (AABBDD), the Zn efficiency was 60%, 36% and 64% respectively. This variation was attributed to the different ABD genome components. The A and D genomes were reported to contribute to high Zn efficiency.

Morgounov et al. (2007) reported grain Fe concentration in the range of 39-48 mg/kg in a set of 25 spring wheat cultivars and 34-43 mg/kg for 41 winter wheat cultivars with the exception of one spring wheat cultivar, Chelyaba, that had grain Fe concentration of 56 mg/kg. Similarly, the Zn concentrations reported in the winter wheat and the spring wheat sets were 23–33 and 20-39 mg/kg, respectively (Tiwari et al., 2009).

During the past decades, the Consultative Group on International Agricultural Research (CGIAR) has taken a major initiative in the program "Harvest-Plus". International Maize and Wheat Improvement Center (CIMMYT) have initiated research to determine the available genetic variability for grain mineral elements from a wide range of germplasm in the genus Triticum, and to study the feasibility of using them in future wheat-breeding programs. Under HarvestPlus program, more than 3000 wheat accessions were screened for zinc and iron concentration by the International Maize and Wheat Improvement Center (CIMMYT). Zinc concentration ranged from 20-115 ppm and iron concentration was recorded in the ranges of 23-88 ppm with the highest levels found in landraces. The most promising germplasms were evaluated across the sites and generations and it was verified with respect to stablility. While variances were associated with environmental effects, high heritabilities were observed for zinc and iron concentrations across environments. Breeding efforts are being done on transferring the zinc trait from diverse sources into locally adapted, agronomically competitive germplasm, considering consumer preferred end-use quality attributes. (HarvestPlus, 2014).

Genetic basis of accumulation of micronutrients

Several studies have been done to understand the genetic basis of accumulation of micronutrients in the grain. Identification and mapping of QTL have been performed to improve grain quality through marker-assisted selection.

Joppa et al. (1997) identified a major QTL Gpc-B1 from wild emmer wheat (Triticum turgidum ssp. dicoccoides) and mapped on chromosome arm 6BS. Later on, it was cloned and confirmed to be effective in improving Fe, Zn, and protein concentrations by 18%, 12%, and 38%, respectively (Uauy et al., 2006). Distelfeld et al. (2006) reported a molecular marker Xuhw89  tightly linked to the Gpc-B1 locus.

Stangoulis et al. (2007) mapped three QTLs for grain Fe accumulation in rice on chromosomes 2S, 8L, and 12L and 2 QTL for grain Zn on chromosomes 1lL and 12L. Tiwari et al., 2009 reported the QTL for grain Fe and Zn in RIL population derived from T. boeoticum accession pau5088/T. monococcum accession pau14087. The grain Fe and Zn concentrations in the RIL population ranged from 17.8 to 69.7 and 19.9 to 64.2 mg/kg, respectively. The QTL analysis led to the identification of 2 QTL for grain Fe on chromosomes 2A and 7A and 1 QTL for grain Zn on chromosome 7A. The region where grain Zn QTL was mapped on rice chromosome 1 is orthologous to wheat chromosome 7.

Genc et al., 2009 identified four QTL associated with grain Zn concentration in a double haploid population derived from Zn inefficient and moderately Zn efficient genotype. These were located on chromosomes 3D, 4B, 6B and 7A and they described 92% of the genetic variation. Each QTL had a relatively small effect on grain Zn concentration but combining the four high Zn alleles increased the grain Zn by 23%.

Xu et al. (2012) analyzed the grain Zn, Fe and protein concentrations in a recombinant inbred line (RIL) population under two environments to detect quantitative trait loci (QTLs) and their interactions. They identified nine additive and four epistatic QTLs, among which six and four, respectively, were effective at both the environments. Three intervals that affected two or three traits were found on chromosomes 4B and 5A, indicating a common genetic basis among grain Zn, Fe and protein concentrations.

Peleg et al., 2008a, b studied the micro and macronutrient content in tetraploid wheat population of 152 recombinant inbred lines (RILs), derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Wide genetic variation was evident among the RILs for all grain minerals. Most QTLs out of 82 were in favor of the wild allele (50 QTLs). Fourteen pairs of QTLs for the same trait were mapped to seemingly homoeologous positions, which reflected synteny between the A and B genomes. Chromosomes 2A, 5A, 6B and 7A were found to harbor clusters of QTLs for grain protein concentration and other nutrients.

However, most of the QTL mapping studies performed until now have only addressed variation for accumulation of iron and zinc in whole-grains. QTL mapping has been done to identify the QTLs controlling variation for the accumulation of micronutrients in the endosperm of cereals. It is an important factor because more than 75% of minerals accumulated in whole grain are lost during milling and micronutrient accumulation is significantly affected by the environment. Thus, painstaking efforts are required to map QTLs controlling iron and zinc accumulation in endosperm of cereal grains, and to identify the genetic determinants and developing markers, which may be used in various genetic backgrounds and under different environmental conditions. An important mechanism that may be exploited in future is transgression. In many QTL studies, positive transgressive segregates accumulated about 2-fold higher iron and zinc than the highest parent. It indicates the possibility of increasing the micronutrient concentration more than the parent.

Challenges ahead

The genotype and environment interaction with respect to grain yield and nutrient density has not been clearly understood. Most of the studies for improvements in nutrient use efficiency have been limited by expensive and laborious phenotyping. Furthermore, bioavailability of nutrients is another important factor in assessing the grain quality. Changing climate conditions may further exaggerate the problem. It was reported that C3 grains and legumes have lower concentration of Zn and Fe, when grown under field conditions at the elevated atmospheric CO2 concentration. Myers et al., 2014 reported that wheat grown at elevated CO2 had 9.3% lower Zn and 5.1% lower Fe as compared to those grown at ambient CO2. Drive for higher yield is generally accompanied with the dilution effect of minerals due to more starch accumulation in grains. Therefore, more focused efforts are required to achieve the goal.

References

Borg S, Brinch-Pedersen H, Tauris B, Madsen LH, Darbani B, Noeparvar S et al. (2012) Wheat ferritins: improving their on content of the wheat grain. J Cereal Sci 56: 204–213.

Bouis HE, Welch RM (2010) Biofortification - A sustainable agricultural strategy for reducing micronutrient malnutrition in the Global South. Crop Sci 50: S20-S32.

Cakmak I, Tolay I, Ozdemir A, Ozkan H, Ozturk L, Kling CI (1999) Differences in zinc efficiency among and within diploid, tetraploid and hexaploid wheats. Ann Bot 84:163-171.

Chatzav M, Peleg Z, Ozturk L, Yazici A, Fahima T, Cakmak I, Saranga Y (2010) Genetic diversity of grain nutrients in wild emmer wheat: potential for wheat improvement. Ann Bot 105 (7): 1211-1220

Deinlein U, Weber M, Schmidt H, Rensch S, Trampczynska A, Hansen TH, et al. (2012) Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation. Plant Cell 24: 708–723.

Distelfeld A, Uauy C, Fahima T, Dubcovsky J (2006) Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker. New Phytol 169: 753–763.

Erdal I, Yilmaz A, Taban S, Eker S, Torun B, Cakmak I (2002) Phytic acid and phosphorus concentrations in seeds of wheat cultivars grown with and without zinc fertilization. J Plant Nutr 25:113-127.

Genc Y, Verbyla AP, Torun AA, Cakmak I, Willsmore K, Wallwork H, McDonald GK.(2009) Quantitative trait loci analysis of zinc efficiency and grain zinc concentration in wheat using whole genome average interval mapping. Plant Soil 314:49–66.   

Gomez-Galera S, Rojas E, Sudhakar D, Zhu CF, Pelacho AM, Capell T, et al. (2010) Critical evaluation of strategies for mineral fortification of staple food crops. Transgenic Res 19: 165–180.

Guttieri MJ, Peterson KM, Souza EJ (2006) Agronomic performance of low phytic acid wheat, Crop Sci 46:2623-2629.

HarvestPlus (2014) Biofortification progress briefs. www.HarvestPlus.org

Joppa LR, Du CH, Hart GE, Hareland GA (1997) Mapping gene(s) for grain protein in tetraploid wheat (Triticum turgidum L.) using a population of recombinant inbred chromosome lines. Crop Sci 37: 1586–1589.

Liu ZH, Wang HY, Zhang GP, Chen PD, Liu DJ (2006) Genotypic and spike positional difference in grain phytase activity, phytate, inorganic phosphorus, iron and zinc contents in wheat (Triticum aestivum L.). Cereal Sci 44: 212-219.

Lu L, Tian S, Zhang J, Yang X, Labavitch JM, Webb SM et al. (2013) Efficient xylem transport and phloem remobilization of Zn in the hyperaccumulator plant species Sedum alfredii. New Phytol 198: 721–731.

Morgounov A, Gomez-Becerra HF, Abugalieva A, Dzhunusova M, Yessimbekova M, Muminjanov H, Zelenskiy Y, Ozturk L, Cakmak I (2007) Iron and zinc grain density in common wheat grown in Central Asia. Euphytica 155:193-203.

Murray CJL, Lopez AD (2013. Measuring the global burden of disease. N Engl J Med 369: 448–457.

Myers SS, Zanobetti A, Kloog I, Huybers P, Leakey ADB, Bloom AJ, Carlisle E, Dietterich LH, Fitzgerald G, Hasegawa T et al. (2014) Increasing CO2 threatens human nutrition. Doi: 10.1038/nature13179.

Peleg Z, Saranga Y, Yazici A, Fahima T, Ozturk L, Cakmak I (2008a) Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat under contrasting irrigation regimes. Plant Soil 306: 57-67.

Peleg Z, Saranga Y, Suprunova T, Ronin YI, Roder MS, Kilian A, Korol AB, Fahima T (2008b) High density genetic map of durum wheat x wild emner wheat based on SSR and Dart markers. Theor Appl Genet 117:103-115.

Raboy V, Noaman MH, Taylor GA, Pickett SG (1991) Grain phytic acid and protein are highly correlated in winter wheat. Crop Sci 31:631-635.

Rellán-Álvarez R, Giner-Martínez-Sierra J, Orduna J, Orera I, Rodríguez- Castrillón JÁ, García-Alonso JI etal (2010) Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato re supplied with iron: new insights into plant iron long-distance transport. Plant Cell Physiol 51: 91–102.

Schlegel R, Cakmak I, Torun B, Eker S, Tolay I, Ekiz H, Kalayi M, Braun HJ (1998) Screening for zinc efficiency among wheat relatives and their utilization for alien gene transfer. Euphytica 100:281-286.

Sperotto RA, Ricachenevsky FK, Waldow VD, Fett JP (2012) Iron biofortification in rice: it’s a long way to the top. Plant Sci 190: 24–39.

Stangoulis JCR, Huynh BL, Welch RM, Choi EY, Graham RD (2007) Quantitative trait loci for phytate in rice grain and their relationship with grain micronutrient content. Euphytica 154:289-294.

Tauris B, Borg S, Gregersen PL and Holm PB (2009) A road map for zinc trafficking in the developing barley grain based on laser capture microdissection and gene expression profiling. J Exp Bot 60: 1333-1347.

Tiwari VK, Rawat N, Chhuneja P, Kumari N, Aggarwal R, Randhawa GS, Dhaliwal HS, Keller B and Singh K (2009) Mapping of Quantitative Trait Loci for Grain Iron and Zinc Concentration in Diploid A Genome Wheat. J Hered 100(6):771–776.

Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314: 1298–1301.

Wang S, Yin L, Tanaka H, Tanaka K, Tsujimoto H (2011) Wheat- Aegilops chromosome addition lines showing high iron and zinc contents in grains. Breeding Sci 61:189- 195.

Welch RM, Graham RD (2004) Breeding for micronutrients in staple food crops from a human nutrition perspective. J Exp Bo. 55: 353-364.

Wessells KR, Brown KH (2012) Estimating the global prevalence of zinc deficiency:results based on zinc availability in national food supplies and the prevalence of stunting. PLoSONE 7:e50568.

White PJ, Broadley MR (2005) Biofortifying crops with essential mineral elements. Trends Plants Sci 10: 586-593.

WHO (2008) World wide Prevalence of Anaemia 1993–2005:WHO Global Database on Anaemia, eds B. de Benoist, E.McLean, I. Egli, and M. Cogswell (Geneva: World Health Organization Press).

WHO (2009) Global Health Risks. Mortality and Burden of Disease Attributable to Selected Major Risks. Available at: http://www.who.int/healthinfo/global_burden_ disease/ GlobalHealthRisks_report_annex.pdf.

Xu YF, An DG, Liu DC, Zhang AM, Xu HX, Li B (2012) Mapping QTL epistatic effects and QTL x treatment interactions for salt tolerance at seedling stage of wheat. Euphytica 186: 233-245.