Zinc biofortification as an innovative technology to alleviate the zinc deficiency in human health: a review

Paddy-wheat crop rotation is responsible for declining soil health, underground water table, arising new micronutrient deficiencies, new weed flora, and resistance to herbicides, declining both land and water productivity and is claimed to be capital and energy-intensive, more particularly in emerging countries. This is further aggravated when micronutrients are deficient, particularly zinc (Zn), which plays an important role in human health, especially in developing countries. Zn biofortification is a technique in which the inherent Zn status of the edible portion of plants is improved by simply spraying a Zn solution onto the crop or through a soil application at a predetermined stage and a proper dose. The concentration of Zn within a wheat grain is genotype-dependent and interacts with the environment, inducing variation in a grain’s concentration of micronutrients. Grain quality parameters are positively correlated with a higher dose of nitrogen in the late reproductive stage. Broadcasting of ZnSO4·7H2O at 62.5 kgha and foliar application of Zn chelates such as Zn–HEDP (C) at 2 g L, Zn–HEDP (L) at 3 g L, or a 0.4–0.5% ZnSO4 solution during grain development stage enhanced the growth, productivity, and micronutrients concentration in the edible portion of the plant which further improves the quality of wheat grains and ultimately improves human health in the region. Given the central importance to Zn in cereal-based nutrition, zinc biofortification appears as an innovative technology to alleviate the zinc deficiency in human health, especially on the Indian subcontinent, by applying Zn either as a foliar or soil application.


Introduction
The rising micronutrient deficiencies more particularly of zinc (Zn) is claimed to be the major reason for the declining land and water productivity of both rice and wheat yield in South Asia Human dependence upon cereals with a poor Zn status, especially in developing countries, deepens the gap between the available amount, and the amount required for good health, which is 40-50 ppm (Cakmak 2010). In India, Zn availability in wheat cultivars varies from 20 to 30 ppm (Shukla et al. 2014). Second, the inherent Zn capacity of the soils decides Zn in wheat grains (Cakmak and Kutman 2018; Figure 1). Hence, if Zn-deficient soils are used for cultivating cereals, then their availability in the grain is decreased to many folds. Therefore, it is essential to sustain a satisfactory level of Zn and water in the soil during the reproductive stage of wheat to improve the Zn status in wheat grains (Cakmak and Kutman 2018). On the other hand, Zn is generally found in excess in the aleurone and embryo (100 ppm of Zn) of a wheat grain whereas white flour, which is derived from the endosperm, contains about 5-10 ppm. When wheat flour is milled, Zn-rich parts (i.e., the aleurone and embryo) are mostly removed and only the endosperm (Zn-poor; about 5-10 mg Zn kg −1 ) remains, making wheat flour Zn-poor (Ozturk et al. 2006; Cakmak and Kutman 2018; Figure 2).
In addition to these factors, the "dilution effect" is responsible for decreased Zn content in the edible portion of cereals with significantly increased production (Shewry et al. 2016). Further, when soil pH exceeds 7.8, Zn particles adhere to clay sites more strongly and hence making it difficult to fill soil solution, causing Zn deficiency (Dotaniya and Meena 2015;Goulding 2016). Diffusion is mainly responsible for the inward movement of the Zn particles in roots, which is severely restricted in the soils equipped with lower organic matter and lower moisture regimes (Rengel 2015).
More than three billion people worldwide are affected by zinc deficiency . Human health complications such as stunting, infections, impaired brain function, poor mental development, weakness in babies and anemia are because of zinc deficiency (Fraga 2005;Cakmak et al. 2010). Further around 200 enzymes responsible for growth, development, immune function, and resistance to infections are regulated by zinc (Fischer and Black 2004). For fulfilling the daily calorie intake, wheat is an important cereal crop and enhancing its nutritional quality will certainly improve the consumer's health (Cakmak 2008).
Institute of Medicine, Food and Nutrition Board (IMFN) reviews and finalizes the daily limits of Zn and other micronutrients that must be consumed or dietary reference intake (DRI) for a healthy life ( Table 1; IMFN  2001) which changes as per variation in age and gender ( Table 1; IMFN 2001). However, in poor or developing countries, hunger can arise from the lack of vitamins and/or mineral elements (Müller and Krawinkel 2005). One way to address the latter is by frequently eating fish and animal products, although poverty and religious backgrounds would be a financial limiting factor. Further, Zn-deficient rhizosphere complicates the situations as products are Zn deficient too (Welch and Graham 2005). In such a case, biofortification could be  a suitable solution, although strategies to increase mineral intake through a diet depend upon many factors and, therefore, might not be successful. Crop biofortification is therefore recommended for directly satisfying the plants' needs to produce the healthy edible portion. The wheat crop is estimated to remove 66-209 g of Zn for every 2 tons of wheat grains. Micronutrient analysis of soil samples across Indo-Gangetic plains revealed that 45.4% of soil was deficient in Zn (Singh and Yadav 2006), as much as 48% of soil in India is deficient in Zn Despite the latest breeding advances in improving nutrient uptake efficiency to cereal grains, these newly bred varieties are unable to fortify the Zn content of cereals in zincdeficient soils (Ortiz-Monasterio et al. 2011). The higher concentration of minerals may be restricted as their supply is governed by different physicochemical properties of soils, which if adversely effected then certainly restrict the micronutrient supply (Frossard et al. 2000). Problematic soils and arid and semi-arid environments mainly magnify zinc deficiency. Zinc deficiency significantly appeared in soils of India (50%), Turkey (50%), China (0.33%), and Western Australia (Ismail et al. 2007), since diffusion and the rate of translocation of Zn are the major limiting factors for the lesser Zn availability in soils for absorption by plants. Therefore, spraying zinc solution or biofortification is a suitable and reliable answer to improve zinc status and, hence, the quality of the produced grains. Zinc is consumed across the plasma, which covers root cells as Zn 2+ (Ismail et al. 2007) while zinc is also permeable to plasma membrane Ca 2+ channels (White et al. 2002a). As the cell proteins are generally bonded by available Zn 2+ , which ultimately reflects on the cytoplasmic Zn 2+ concentrations (Broadley et al. 2007). In such cases, Zn has to be applied from the outside, either as a foliar spray or soil application, to attempt to enhance Zn availability in grains. Given the central importance of Zn in cereal-based nutrition, especially on the Indian subcontinent, this review aims to assess Zn biofortification as a way to alleviate Zn deficiency in human health.

Physiological basis of agronomic biofortification
Agronomic biofortification allows for mineral density to be increased in grains or fruits through fertilization strategies at responsive growth stages of crop plants . Mineral supply to a developing cereal grain takes place either by direct uptake from the soil or by remobilization of stored minerals in leaves. Nutrient density per unit of grain dry weight is more important for estimating grains' quality (Marles 2017). At critical growth stages of a crop, proper supply of micronutrients improves not only the quality of grains but also the health status of ultimate consumersthe human (Marschner 1995). However, oversupply or limiting micronutrients can have negative consequences. Mineral enrichment occurs when a nutrient exceeds the level of sufficiency within a crop plant ( Figure 3). Through leaves, plants have the capability to absorb the different nutrients; therefore, foliar spray of micronutrients would theoretically imply that an applied nutrient will be absorbed from leaves to the point of utilization, viz., growing tissue (Khoshgoftarmanesh et al. 2010). Nutrients are exported from leaves and transported within the stem via phloem or xylem (Rengel et al. 1999). The biofortification of micronutrients at specific and critical growth stages of wheat may contribute toward grain mineral enrichment and enhanced yield by improving their availability (Marschner 1995). Application of the zinc either through soil or through foliar application is reported as an effective method for improving grain Zn concentration (Cakmak 2010). The timing of a micronutrient foliar spray is an important parameter that delineates its effectiveness in increasing its content in grain. For instance, significant land productivity increase is most likely with foliar application of Zn fertilizer

Zn transport mechanism in plants
Nutrients need to move through simplistic cells of the plant before reaching to the grains. Generally, two methods are used by the plant roots for making metal ions more available for uptake ( Figure 4). Foremost, soil inherent fertility decides the roots acidification of the rhizosphere through plasma membrane H + -ATPase.

Significance of biofortification
The biofortification technique for grain quality improvement is practiced for popular wheat cultivars. This approach is beneficial to the farmers because they get  nutrient-fortified seed which is in high demand in the local market for overall improving their livelihood. The farmer is protected from buying new seeds and investment in micronutrient fertilizers is saved. Improvements in micronutrient concentration are associated with an increase in land productivity. The application rates of micronutrient fertilizers applied as a foliar spray are much smaller. Hence, this technique is a win-win approach for all. 6 Review of previous research to assess research gaps The nutrient density of seed is dependent on inherent fertility status, soil type, crop species, season, and cultivars (Ascher 1994). Different genotypes may differ in phonological behavior and interaction with diverse management practices due to genetic variation. Varieties with heights have lesser both land and water productivity than the dwarf ones as lesser responsive to applied fertilizers. Dry matter accumulation and yield attributing characters mostly differ when different cultivars were selected based on their genotypic sequencing. Being greater in the efficient use of nitrogen fertilizer and resistant to lodging, dwarf cultivars were responsible for the green revolution in the region. In research trials at PAU Ludhiana, wheat genotypes, viz., HD 2329 and WH 542 showed significantly higher plant production parameters than durum wheat (Singh et al. 1996). Hence, the varietal performance may change according to the genetic constitution and the agroclimatic conditions in the ambiance. In an accession from Spain "ANDALUCIA 344," highest levels of Zn concentrations were reported. Significant and positive correlation was reported between Zn and Fe concentrations (r = 0.81; p < 0.01), in "HONG DUAN MANG" (Chinese spring bread wheat). Therefore, cultivar selection is a must step to consider not only to maintain but also to improve the Zn-use efficiency, which further needs to consider the climate, soil type, organic matter content, and inherent fertility of the soil.  Kumar and Ahlawat (2006) showing significantly higher N uptake in wheat-maize cropping system with 120 kg N ha −1 than 0 and 60 kg N ha −1 . Sedimentation value determines the functional quality of gluten proteins present in the grain. However, nitrogen has a non-significant effect on the sedimentation value of wheat grains in sandy loam soils ). The grain appearance score depends upon grain-size, shape, luster and color. Grains are uniform in shape, bold in size, glossy in luster, and amber in color due to significantly their higher protein and beta-carotene content. Hardness and virtuousness are mechanical and optical properties, respectively. Vitreous endosperms have high gliadin content that causes higher adhesion of the protein matrix on starch granules during kernel desiccation causing compact endosperm shape (Samson et al. 2005). The application of 150 kg N ha −1 recorded maximum nitrogen uptake over control in wheat (Singh and Yadav 2006). The maximum protein content (12.3%) was found at 180 kg N ha −1 on loamy sand soils ). An attempt to improve the functional quality of indigenous wheat was made by Anureet et al.

Growth parameters and grain yield
Applying Zn fertilizers to wheat resulted in improved grain quality and higher land and water productivity . Therefore, Zn alone or in combination with N not only improves growth parameters but also improves land productivity. Applying Zn fertilizers to wheat grown in fields in central Anatolia, Turkey, improved grain Zn concentration (Yilmaz et al. 1997). An increased Zn transport from leaves into seeds can be achieved by spraying Zn solution of 0.5%, particularly under environmental stress conditions (e.g., drought) and on potentially deficient Zn soils (Yilmaz et al. 1997). The higher increase in the percentage of protein could easily be achieved through the soil and foliar application of Zn fertilizers (Ranjbar and Bahrmaniar 2007). Therefore, the role of the agricultural scientists cannot be ignored as far as biofortification or enriching edible portion of plants with micronutrients, viz., Zn is concerned. Furthermore, the foliar Zn application doubled grain Zn concentration (Peck et al. 2008). It was also reported by Cakmak (2008) that a 3.5-fold hike in the grain Zn concentration could be achieved by zinc biofortification. The boost in Zn percentage through the foliar application over non-foliar application was more in Zndeficient soils as compared to Zn-sufficient soils (Ram et al. 2011). Foliar Zn application was much more effective than soil Zn application in the enrichment of wheat grains. The foliar 0.4% ZnSO 4 ·7H 2 O application resulted in the best effect on grain Zn with 58% increase in grain Zn concentration, 76% increase in wheat flour Zn, and up to 50% decrease in the molar ratio of phytic acid to Zn in flour (Zhang et al. 2011). At the early grain development stage, Zn solution (0.5%) spray considerably improved Zn concentration in edible seeds and decreased concentration of phytic acid and ratio of phytic acid to Zn molar (Xi-wen et al. 2011). Zn application combined with foliar spray during the grain development stage increased grain Zn concentration by 95% and whole-grain estimated bioavailability by 74% . For ameliorating the Zn deficiency and prevent yield losses in cereals, Zn needs to be applied to deficient soil, typically in the form of ZnSO 4 , at rates that range typically from 50 to 62.5 kg Zn ha −1 . Several factors, viz., inherent zinc concentration, soil texture and method of zinc application, and alkaline or calcareous soils, affect zinc biofortification (Alloway 2009). Zn fertilization has residual effects for up to 10 years and is not needed every year as only a small amount is being taken up by crops every year (Shivay et al. 2008). Therefore, zinc biofortification does not need to be carried out every year but even then farmers have to keep a watch on the crop stand, and it should be applied based on the appearance of its deficiency symptoms or on the basis of soil test reports.

Conclusion
Zinc-biofortification of crops either by soil or by the foliar method is required in the present era of intensive agriculture. Mineral fertilizers both macro and micro combined with proper soil fertilization approaches with an increased ability to improve the zinc density of grains, are advocated. Humans in both poor/developing countries, viz., India will accept biofortified grains if not more expensive than nonfortified grains as they are almost similar in appearance, taste, texture, or cooking quality of foods (Bouis 2003). Biofortified crops will have a great demand if their beneficial aspects to human health are demonstrated to consumers. Certainly, biofortified crops along with targeted genetic manipulation show great potential to address hidden hunger in humans across the world. However, there is a need to check the extent of increasing mineral density throughout the world in texturally divergent soils under different climatic conditions and different eating food habits of the inhabitants. Furthermore, there is a need to revise our old formulated fertilizer recommendations keeping in view the present trends of micronutrient deficiencies more particularly of Zn for the overall improvement of the health status of poor/developing nations and to get rid of the "Hidden Hunger" for the inclusive betterment of human beings.
Funding: The review was developed in the absence of financial support.
Author contributor: RH and PS prepared the initial content and draft of the review; AH co-wrote and edited all versions of the manuscript and also submitted to the journal as a corresponding author for publication. All authors have seen and approved the final version of the review for publication in the journal.

Conflicts of interest:
The authors declare no conflict of interest.
Informed consent: Informed consent was obtained from all individual co-authors.