Advanced biotechnological strategies towards the development of crops with enhanced micronutrient content

Abstract

Micronutrients are essential mineral elements required for both plant and human development.An integrated system involving soil, climatic conditions, and types of crop plants determines the level of micronutrient acquisition and utilization. Most of the staple food crops consumed globally predominantly include the cereal grains, tubers and roots, respectively and in many cases, particularly in the resource-poor countries they are grown in nutrient-deficient soils. These situations frequently lead to micronutrient deficiency in crops. Moreover, crop plants with micronutrient deficiency also show high level of susceptibility to various abiotic and biotic stress factors. Apart from this, climate change and soil pollution severely affect the accumulation of micronutrients, such as zinc (Zn), iron (Fe), selenium (Se), manganese (Mn), and copper (Cu) in food crops. Therefore, overcoming the issue of micronutrient deficiency in staple crops and to achieve the adequate level of food production with enriched nutrient value is one of the major global challenges at present. Conventional breeding approaches are not adequate to feed the increasing global population with nutrient-rich staple food crops. To address these issues, alongside traditional approaches, genetic modification strategies have been adopted during the past couple of years in order to enhance the transport, production, enrichment and bioavailability of micronutrients in staple crops. Recent advances in agricultural biotechnology and genome editing approaches have shown promising response in the development of micronutrient enriched biofortified crops. This review highlights the current advancement of our knowledge on the possible implications of various biotechnological tools for the enrichment and enhancement of bioavailability of micronutrients in crops.

Introduction

Currently, micronutrient deficiency or hidden hunger is considered as one of the major global issues. It affects nearly two billion people, and the cases are predominantly found in Asia, Latin America and Africa (Pfeiffer and McClafferty 2007).In various countries, human micronutrient deficiency is a major concern as the staple foods, including tubers, roots, and cereals are grown in nutrient-poor soils (Saltzman et al. 2014). Along with the production of nutrient rich crops, achieving the goal of an adequate amount of food production is one of the major challengesto cope up with the need of the increasing global population. To fulfill this aim of improved crop yield, improvement in the quality and quantity of crop production is crucial (Tilman et al. 2011). Besides these issues, due to climate change, plants are also facing various forms of abiotic stresses (drought, salinity, flood, extremely high or low temperature, UV radiation) and challenges from biotic factors (fungi, nematodes, bacteria, herbivores, and different types of insects), leading to decreased crop production globally (Parry and Hawkesford 2010).For optimal growth and development, plants require some macronutrients and micronutrients. Nitrogen (N), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg), and Sulphur (S) are known as essential nutrients, which are required in large quantities, thus commonly known as macronutrients. A distinct group of other essential elements, such as Copper (Cu), Iron (Fe), Manganese (Mn), Molybdenum (Mo), Chlorine (Cl), Nickel (Ni), Boron(B), and Zinc (Zn) are required in lesser amounts and are collectively called as micronutrients (White and Brown 2010; Kabir 2016; Kumar et al. 2018). Some other elements, like Selenium (Se), Sodium (Na), and Silicon (Si) are also required in very low amounts to enhance the growth and developmental response in plants. These elements are known as beneficial elements (Kabiri et al. 2017).

In humans, the most frequent micronutrient deficiencies are associated with Iron (Fe), Zinc (Zn), and Iodine (I), respectively (Saltzman et al. 2014). Previously, several studies have shown that micronutrient deficiencies cause detrimental effects and health problems, such as impaired cognitive development, perinatal complications, and premature death, respectively (Bailey et al. 2015).A deficiency of any one of the essential macronutrients or micronutrients adversely affects plant growth and development and eventually crop yield. Although, increased use of chemical fertilizers enhances crop production, it also causes severe damage to the agricultural land, including a reduction of soil structure and fertility, which finally leads to mineral imbalance in the soil. In general, the nutrients required by the plants are incorporated into the fertilizers so that plants can easily absorb them from the soil. In recent studies, it has been found that despite the presence of sufficient levels of various micronutrients, plants cannot absorb all the micronutrients efficiently. Therefore, it is an important aspect to explore the appropriate strategies to enhance the nutrient uptake efficiency in plants (Datnoff et al. 2007).

Micronutrient deficiency has either direct or indirect effects on the vulnerability of plants to various environmental stresses (Hajiboland 2012). Plants, which grow under micronutrient deficient conditions, are more susceptible to various abiotic and bioticstresses (Ahmad and Prasad 2011). Therefore, along with the enhancement of the crop and seeds’ nutritional value, additional challenges include the judicious use of chemical fertilizers and development of stress tolerance in crops. Diverse methods and techniques have been suggested and implemented for the past couple of years to overcome the problem of micronutrient deficiencies in the staple crops. Despite some limitations, biofortification method has received considerable attention during the recent years for the improvements of micronutrient deficiencies in crops (De Steur et al. 2015). The growing process of enrichments ofmicronutrients and other essential nutrients in food crops through several techniques, such as conventional breeding, agricultural biotechnology andadvanced agronomic practices is collectively known as biofortification (Bouis and Welch 2010; Bouis and Saltzman 2017; Garg et al. 2018). On the other hand, nanotechnology mediatedimprovement of plant nutrient uptake efficiency has been demonstrated in some recent studies to add value to agriculture using nanoscale properties (Thakur et al. 2018; Nile et al. 2020; Kalra et al. 2020).

Significant advancements have been made during the last couple of years in plant breeding approaches to unfold many plant genotypes with enhanced nutrient uptake efficiency.Recent advances in plant biotechnology technologies, such as the genome-editing approaches, including the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), transcription activator-like effector nuclease (TALEN), and the associated extensive studieson genetic diversity in crops and other plant species have facilitated in the development of biofortified crops (Díaz-Gómez et al. 2017).In additional, several genetic engineering techniques have also been employed to increase the micronutrient levels and eliminate anti-nutrient substances (such as phytic acids) in plants (Li et al. 2012). Recently, many transgenic plants with maximized amounts of vitamin A, S-containing amino acids, and the elements of Fe and Zn have been developed(Hefferon 2020).During the recent years, transgenic approaches have also been utilized in a more selective way for the introduction of desirable traits in crop plants and to improve nutrient uptake efficiency (Garg et al. 2018).

Plant growth response depends on micronutrients

Maintenance of global food and nutrition security is considered as one of the greatest challenges under the changing climatic condition. Approximately, over half of the world’s population is malnourished to date,and is a matter of serious concern. Rice, wheat, and maize are the major source of global food supply and representing about 90% of cereal production worldwide.The human population largely depends on these major cereal crops for the supply of food and nutrition (Sandhu et al. 2021). Therefore, to feed and provide nutrition to the increasing population, plant biologists are continuously working on the improvement of nutritional value, productivity, sustainability, and environmental protection of the cereal crops as well as natural plant species (Sandhu et al. 2021). Plant growth and development are largely dependent on the mineral nutrients present in the rhizospheric soil. There are at least seventeen essential elements, which are required for maintaining plant growth and development. These essential elements are broadly classified as non-mineral elements, macronutrients, and micronutrients, respectively.Micronutrients, including B,Cl,Cu, Fe,Mn, Mo, Zn,and Ni are required in very low concentrations (5-100 mg/Kg dry weight) and their deficiency inhibit plant growth and development (White and Brown 2010; Kabir 2014; Kumar et al. 2018) (Table 1). Apart from micronutrient efficiency, low level of uptake of some micronutrients in some crops is also another important issue and therefore, in recent years, emphasis has also been given by the plant breeders on the improvement of nutrient uptake efficiency in crops. Considerable advancements have been made to implement various strategies for enhancing crop’s nutrient use efficiency and to reduce the use of fertilizers and other agrochemicals in the agricultural field.

Table 1.

Different membrane-bound transporters associated with micronutrient uptake and micronutrient deficiency symptoms in plants

Micronutrients	Transporters	Deficiency symptoms in plants	Reference
Zinc (Zn)	1. Zinc-iron permease or ZRT/IRT like protein (ZIP) 2. Heavy metal ATPase (HMAs) 3. Metal tolerance protein (MTPs) 4. Natural Resistance-Associated Macrophage Protein (NRAMP) and 5. Vacuolar Iron Transporter (VIT)	Inhibition of internodal elongation. Inhibition of root growth and leaf size. Leaf shows chlorotic and necrotic spots.	Fageria et al. 2008; Moreira et al. 2008; Zhang et al. 2012; Olsen and Palmgren 2014
Iron (Fe)	1. Plasma membrane proton pump P-type ATPase 2. Ferric reductase oxidase family enzyme FeIII 3. Iron regulated transporter (IRT) transport protein of the Zinc regulated transporter (ZRT) IRT like protein (ZIP) family	Decreased plant growth and crop productivity.	Kobayashi and Nishizawa 2012; Sperotto et al. 2012
Copper (Cu)	High-affinity COPT transporters	Delayed flowering and reduction in pollen viability.	Yamasaki et al. 2009; Bernal et al. 2012;
Boron (B)	1. Tonoplast intrinsic proteins (TIPs) 2. Plasma-membrane intrinsic protein (PIPs) 3. Small basic intrinsic protein (SIPs) 4. Nodulin 26-like intrinsic proteins (NIPs).	Inhibition of root and shoot growth. Premature flowering and reduced flower production.	Takano et al. 2006; Marschner 2012
Manganese (Mn)	1. Natural Resistance Associated Macrophage Protein (NRAMP) family 2. Zinc Regulated Transporter/Iron-Regulated Transporter (ZRT/IRT)- related Protein (ZIP) family 3. Yellow Stripe-Like (YSL) family. 4. Cation Diffusion Facilitator/Metal Transport Protein (CDF/MTP) family 5.Vacuolar Iron Transporter (VIT) family 6. Ca2+/Cation Antiporter (CaCA) superfamily 7. Bivalent Cation Transporter (BICAT) family	Reduction in photosynthesis rate. Degradation of chlorophyll. Leaf shows pale mottled symptoms with interveinal chlorosis. Imbalance in plant water relations.	Nevo and Nelson 2006; Broadley et al. 2012; Hajiboland, 2012; Schmidt et al. 2016;
Nickel (Ni)	Active transport and passive diffusion	Reduction in nitrogen metabolism. Necrosis and chlorosis in leaves.	Freitas et al. 2018
Molybdenum (Mo)	MOT1 and MOT2 carriers	Abnormal leaf morphology and poor growth.	Rana et al. 2020
Chlorine (Cl)	Symplastic transport	Wilting of leaves.	Geilfus 2018

Micronutrients have different functions in plants. They are present as co-factors in several plant proteins, such as Fe is present in the reaction center of photosystem and cytochrome. Mn is present in the Photosystem II, while Cu is present in the Plastocyanin (Nikolic and Pavlovic 2018). Moreover, plants contain three types of superoxide dismutase (SOD) such as Fe-SOD, Mn-SOD, and Cu-Zn SOD, and their activitiesare regulated by respective trace elements (Castro et al. 2018). Zn also acts as co-factor in many plant enzymes, including alcohol dehydrogenase, carbonic anhydrase, glutamate dehydrogenase and RNA polymerase, respectively (Moreira et al. 2018). Deficiency in any one of the micronutrients causes severe growth and developmental defects in plants (Table 1). Furthermore, deficiency of many micronutrients is often associated with inhibition of root and shoots growth due tocessation cell elongation (Brown et al. 2002), decrease in pollen viability, pollen infertility, premature flowering, and reduced flower production, respectively (Marschner 2012). Deficiency of boron alters phenolics and nitrogen metabolism by oxidizing phenolic compounds and decreasing nitrate uptake (Camacho-Cristóbal et al. 2002). Symptoms of Mn deficiency first develop in the younger leaves, but the most visible symptoms include the pale mottled leaves with interveinal chlorosis (Broadley et al. 2012; Hajiboland 2012; Schmidt et al. 2016). Moreover, the deficiency symptoms of many micronutrients are more or less similar and sometimes overlapping, such as reduced photosynthetic activity, development of chlorotic and necrotic spots, reduction in grain quality and quantity, abnormal leaf morphology, and wilting of leaves, respectively (Table 1) (Kobayashi and Nishizawa 2012; Hajiboland 2012; Bernal et al. 2012; Olsen and Palmgren 2014; Geilfus 2018).

Different membrane-bound transporter families are involved in uptake of micronutrients

Crops that have higher nutrient use efficiency are characterized by an increased ability of nutrient uptake from the soil from where it is subsequently transported throughout the plant body, eventually determining plant growth and productivity (Ahmad et al. 2021). Several transporter proteins are associated with the uptake and transportation of micronutrients across the cellular membrane in plants (Table 1) (Castro et al. 2018). Plants uptake Zn in two ways,such as the apoplastic and symplastic pathways (Reid et al. 1996). There are three types of transporter families involved in Zn transport in plants, such as Zinc-iron permease or ZRT/IRT like protein (ZIP), which helps in Zn uptake (Fig. 1) from soil to cytosol of root cell, the Heavy metal ATPase (HMAs), associated with xylem loadingvacuolar scavenging and efflux of Zn from plastid, and the Metal tolerance proteins (MTPs), involved in the transportation and accumulation of Zn into the vacuoles (Fig. 1) (White and Broadley 2011; Olsen and Palmgren 2014) (Table 1). Furthermore, some Natural Resistance-Associated Macrophage Protein (NRAMP) and Vacuolar Iron Transporter (VIT), are also involved in Zn transport and homeostasis (Oomen et al. 2009; Song et al. 2010; Zhang et al. 2012).

Fig. 1.

Illustration of the localization and function of essential micronutrient transporters in a plant cell. Uptake of Fe³⁺is facilitated by different transporters such as FRO2, FRO3, FRO4, FRO7, PIC1, VIT1, and NRMP3/4 distributed in different membrane-bound compartments of the cell. Cu²⁺ is transported into the cell by COPT1, COPT2, and ZIP transporters. HMA4 and HMA5 import Cu²⁺ into the vacuole and COPT5 exports Cu²⁺ from the vacuole. ATX1, CCS1, and COX17 are involved in Cu²⁺ delivery to specifically targeted proteins. Zn²⁺ uptake is mediated by ZIP1, ZIP2, ZIP4, and HMA4 transporter proteins. ZIP4 is present in both the chloroplast and mitochondria. MN²⁺ is transported into the cell via YS1, YSL6, NRAMP, and IRTL transporters. MOT1 and MOT2 are involved in Mo import and export to the cell, vacuole, mitochondria, and the endoplasmic reticulum. B is imported and exported within the cell via NIP5;1 and BOR1 respectively. BOR- Borate Transporter; CCS1- Cu⁺ Chaperone for Cu/Zn SOD; COPT- Copper transporter; COX- Cytochrome C Oxidase; ECA1- ER-type Ca²⁺-ATPase1; FRO- Ferric Reductase oxidase; HMA- Heavy Metal ATPase; IRT- Iron Regulated Transporter, MHX- Mg²⁺/H⁺ Exchanger, MIT- Mitochondrial Iron Transporter; MOT- Molybdate Transporter; MTP- Metal Tolerance Protein; NIP- Nodulin 26-like intrinsic protein; NRAMP- Natural Resistance Associated Macrophage Protein; PCR- Plant Cadmium Resistance; PIC- Permease in Chloroplast; RAN1- Responsive to Antagonist 1; VIT-Vacuolar Iron Transporter; YS1- Yellow Stripe 1; YSL- Yellow Stripe-Like; ZAT- Zinc Transporter; ZIP- Zink-iron permease or Zrt/Irt like protein

Plants areunable to take up Fe³⁺ oxides and hydroxides available in the soil. Plasma membrane proton pump P-type ATPase acidifies the rhizosphere or the apoplast by releasing protons and makes Fe³⁺ more soluble for reduction, which is finally reduced by ferric reductase to Fe²⁺. Subsequently, Iron regulated transporter (IRT), ZRT/IRT like protein (ZIP), andYellow Stripe-Like2 (YSL2) transport Fe²⁺ across the plasma membrane (Fig. 1) (Mäser et al. 2001; Vert et al. 2002; Santi and Schmidt 2009; Kobayashi and Nishizawa 2012). Cu is reduced by ferric reductase oxidase 4 (FRO4) and ferric reductase oxidase 5 (FRO5) at the root surface, and then high-affinity transporters, COPT transport the reduced Cu²⁺ ions across the plasma membrane of epidermal cells of root apices (Fig. 1) (Bernal et al. 2012; Garcia-Molina et al. 2013). Moreover, uptake of boron (B) from the soil is achieved in the form of boric acid. Being uncharged, boric acid easily passes through the plasma membrane, though several transporters are present, such as tonoplast intrinsic proteins (TIPs), plasma membrane intrinsic protein (PIPs), small basic intrinsic protein (SIPs), and nodulin 26-like intrinsic proteins (NIP), which helps in the uptake of B in plants (Takano et al. 2006).

Interestingly, plants takeup Mn in the form of Mn²⁺ using various transporters like NRAMP, ZIP, and YSL (Table 1) (Nevo and Nelson 2006).Molybdenum is released from the solid minerals through weathering process. The soluble form of molybdenum, MoO⁴⁻is taken up by themolybdate transporter 1 (MOT1) and molybdate transporter 2 (MOT2) carriers in plants (Kaiser et al. 2005). On the other hand, uptake of nickel occurs in plants via both active transport and passivediffusion system either as Ni²⁺ or as Ni soluble complex (Chatzistathis 2018). Cl⁻ uptake in plants mainly involves the symplastic transport pathway. Cl⁻along with soil water travels into the root and moves into the xylem following a chemical gradient (Gong et al. 2011). Recently, it has been shownthat plant chloride channels (CLCs) transports Cl⁻ and nitrate (NO₃⁻), and play a pivotal role in nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, and signal transduction (Wei et al. 2019).Taken together, this information demonstrates that the transporters play a crucial role in micronutrient uptake and loss of function mutation of any one of them may lead to severe growth defects.

Transcriptional regulation of micronutrient homeostasis

Several studies have demonstrated that transcription factors (TF) play a crucial role in the regulation of many stress-responsive genes (Singh et al. 2002). Moreover, many TFs are directly or indirectly involved in the regulation of micronutrient uptake and accumulation via modulating the expression of many transporter genes in plants (Wang et al. 2013; Liu et al. 2017). The TFs belonging to the B3 family has been shown to play multiple functions in the accumulation of nutrients during seed development (Boulard et al. 2017). Recently, it has also been shown that the B3 class of TFs could be directly or indirectly involved in the Fe uptake in seeds via interacting with the RY motif containing promoters of the genes involved in iron homeostasis (Roschzttardtz et al. 2020). Iron is one of the most important micronutrients in plants because of its involvement in many key cellular functions, such as photosynthesis, respiration and other cellular processes. As mentioned in previous section, deficiency in iron availability seriously affects crop growth and yield quality. It has been shown that in Arabidopsis, 16 bHLH TF regulates the iron-dependent transcriptional regulatory cascade (Gao et al. 2019). In Arabidopsis,four bHLH TFs, namely bHLH34, bHLH104, bHLH105, and bHLH115 interact to form homo or heterodimers and activate plant response to Fe deficiency (Zhang et al. 2015; Li et al. 2016). The OsNAC5 (member of the NAM (no apical meristem), ATAF1-2 (Arabidopsis thaliana activating factor 1–2), and CUC2 (cup-shaped cotyledon 2) superfamily, are possibly involved in Fe and Zn allocation in rice grain (Sperotto et al. 2009). Interestingly, overexpression of OsNAC5 in the flag leaves during grain filling resulted in a higher amount of Fe accumulation in rice seeds (Sperotto et al. 2009). An R2-R3 MYB transcription factor, MdMYB58 in apple was found to be directly involved in the regulation of Fe transport and tissue compartmentalization (Wang et al. 2018). Another study has revealed that WRKY46 plays a crucial role in Fe translocation from root to shoot via repressing the expression of VTL1 (Vacuolar iron transporter-like 1) (Yan et al. 2016; Liu et al. 2017) identified two AP2/ERF TFs, ERF4, and ERF72 that are involved in the inhibition of the expression of two important genes involved in Fe uptake, such as IRT1 and AHA2. The Fe-deficiency Induced Transcription Factor(FIT) forms heterodimer with bHLH38 or bHLH39 and then induces several Fe acquisition genes, including FRO2 and IRT1 (Yuan et al. 2008; Brumbarova et al. 2015). Apart from FIT mediated network, another bHLH TF, POPEYE (PYE) activates additional Fe deficiency-induced genes in Arabidopsis (Long et al. 2010). In rice, an ABI3/VP and NAC/CUP family, iron deficiency responsive element binding factor-1, 2 (IDEF1 and IDEF2), respectively regulate the early response to Fe deficiency genes and distribute Fe through the regulation of the OsYSL2 gene (Kobayashi et al. 2007).

Recently, some F-group b-ZIP TFs have been identified and shown to play animportant role in Zn deficiency response via regulating Zn responsive genes (Lilay et al. 2020). Assuncao et al. (2010)reported two F-group b-ZIP TF, such as b-ZIP19 and b-ZIP23, which play a crucial function in zinc deficiency response in Arabidopsis. The double mutants of bzip19/bzip23 showed hypersensitive response under zinc deficiency (Assuncao et al. 2010a). Moreover, it has been shown that the bZIP-19 and b-ZIP23 can activate the expression nicotinamine synthase (NAS) via binding to its promoter sequence and eventually leads to enhanced uptake of Zn under Zn deficient conditions (Assuncao et al. 2010b). Overexpression of either Cu-responsive TF, such as SPL7 or Cu transport genes, such as COPTshas been shown to be used for the enhancement of Cu content in crop plants (Migocka and Malas 2018).

microRNAs function in micronutrient assimilation

microRNAs (miRNAs) are small non-coding RNAs of about 22 nucleotides long, regulatory RNA molecules, which are involved in almost all metabolic and molecular processes in plants, including regulation of plant growth and development, cell wall biosynthesis, response to various stress conditions and several other processes (Mangrauthia et al. 2017). Several miRNAs are known to play key role in the regulation of uptake and transport of mineral nutrients in plants (Kehr et al. 2013). Recent studies have revealed the involvement of miRNAs in the regulation of phosphate and sulfate-related gene expression in Arabidopsis, rice, and wheat (Pant et al. 2009; Paul et al. 2015). However, the function of miRNAs in the regulation of micronutrient homeostasis in plants is still less explored. Plethoras of studies havedemonstrated downregulation of miRNAsalong withthe upregulation of their target genes involved in micronutrient uptake and homeostasis,indicating their possible involvement in micronutrient homeostasis (Khraiwesh et al. 2012).Previous studies have shown that three miRNAs, namely miRNA169, miRNA395, and miRNA398display reduced expression under Fe deficiency, and also involved in signal transduction pathways of other nutritional deficiency genes. In Arabidopsis, miR159a and miR394a in roots, whilemiR159a and miR169b in shoots showed enhanced expression under Fe deficiency (Kong and Yang 2010). Studies on Fe deficiency response have already revealed that some of the miRNA’s expression is reduced under Fe deficiency, whereas deficiency of Cu increases their expression (Waters and Troupe 2012). Cu plays a crucial role in oxidative stress response in plants via acting as a cofactor of Copper/Zinc Superoxide Dismutase (CSD). During the period of Cu starvation, the induction of miR398 resulted in the reduction in the expression of CSD1,CSD2, and Cu Chaperones for SOD1 (CCS1) (Sunkar et al. 2006; Beauclair et al. 2010). Moreover, it has been shown that the expression of various miRNAs, such as miR397, miR408, and miR857are upregulated under Cu deficiency, eventually resulting in the suppression of different laccase (LAC3, LAC12, and LAC13) and plastocyanin genes (Paul et al. 2015). In Phaseolus vulgaris, different miRNAs, namely miR319, miR169, miR396, miR170, miR164, and miR390have been found to be upregulated under Mn toxicity (Valdes-Lopez et al. 2010). Interestingly, in Sorghum bicolor, Zn deficiencycauses the upregulation of several miRNA families, such as miR166, miR171,miR172, and miR398, miR399, and miR319, modulating the expression of several transporter genes(Li et al. 2013).Taken together, the study of the functional network of miRNAs and their target specificity in the regulation of several genes provides new insights, unveiling the molecular pathways related to micronutrient metabolism. Therefore, miRNAs appears as important genetic targetsfor the identification of novel traits for improvingmicronutrient use efficiency in crops.

Novel genetic approaches to enhance micronutrient uptake efficiency

Accumulation of micronutrients in the soil or edible parts of a plant depends on some rate limiting factors, such as limited mobility of some micronutrients and differences in soil composition (Kalra et al. 2020). Moreover, increasing the concentration of micronutrients via the application of different inorganic fertilizers is quite expensive for the farmers. Besides, excessive application of such chemical fertilizers in the agricultural field is often associated with a reduction in thebiodiversity and enhanced environmental pollution (Jewell et al. 2020).Within this context,development of crops through genetic improvement is considered as more effective approach. Recent advancements on the identification and characterization of genes associated with micronutrient uptake and accumulation haveopenedup an important avenue to combat the challenge of global micronutrient requirements. Traditional genetic approaches related to marker-assisted selection (MAS), genome-wide association study (GWAS), and quantitative trait loci (QTL) mapping emerged as cost-effective techniques for the selection of plants with desired traits. Gene annotation studies have identified OsIRT1 gene as an important candidate for the enhancement of Fe uptake in rice (Bonneau et al. 2018). The genetic basis for the development of biofortified crops depends largely on the QTLs. However, identification of QTLs requirescomprehensive knowledge of a large number of molecular markers and high-resolution genetic maps. Recently, chromosome-based studies haveidentified more than 80 QTLs distributed in 12 chromosomesin riceand found to be involved in Fe and Zn uptake (Sharma et al. 2019). Previously,studies on QTLs led to the identification of ten candidate genes involved in Fe and Zn uptake, transport, and accumulation in rice seeds (Anuradha et al. 2012). Similarly, several QTLs were found to be associated with Mn and Selenium (Se)uptake in rice (Liu et al. 2017; Wang et al. 2017) identified a locus containing the OsNRAMP5 gene,associated withthe function of Mn uptake and accumulation in rice grain. Furthermore, recent studies on DNA single nucleotide polymorphisms (SNPs), simple sequence repeats (SSR) and RNA marker-based marker-assisted selection haveprovided important insights into the development of nutrient-rich maize grain (Masuka et al. 2017). Recently, 20 SNPs have been characterized and found to be directly involved in the accumulation of Zn in maize (Hindu et al. 2018).However, most of theagriculturally important crops are polyploid and with large genome size, thus making them problematic for conventional breeding approaches. In addition, without any prior knowledge of genome sequence, it has been found to be difficult to develop molecular markers and identify QTLs related to desired traits in many other non-conventional crops (Ali and Borrill 2020). Thus, more efficient and advanced strategies are needed to be developed for the improvement of plant micronutrient uptake efficiency.

Biotechnologicaltools used to enhance plant micronutrient use efficiency

The strategies for genetic engineering employed as important biotechnological tools predominantly include mutagenesis, transgenic methods, and genome editing. Transgenic approaches can rapidly introduce desirable traits into different crop varieties and these strategies could be more effective than conventional breeding methods for the enhancement of nutritional value in crops (Fig. 2) (Kumar et al. 2019).The biotechnological approaches are gaining attention because of their less cost and time-intensive nature for the species and targeted manipulation of the desired trait, compared with the traditional genetic approaches.Enhancement of micronutrient content in plants requires the manipulation of the metabolic pathways, which mayeither increase the production of the desirable compounds or decrease the number of competitive compounds or inhibitors (Capell and Christou 2004). Modern genetic and biotechnological tools can be utilized for thedevelopment of biofortified staple foods with a higher micronutrient content and improved bioavailability (Holm et al. 2002).

Fig. 2.

A general view of the strategies and novel technologies of biofortification approaches used to enhance micronutrient use efficiency in plants. Biotechnological approaches can improve plant nutrient uptake efficiency via gene editing, base editing, nanotechnology, and transgenic methods

The approaches to combat micronutrient deficiency and malnourishment have been broadly categorized into three groups (Gómez-Galera et al. 2010). Increased diversity of food intake has been considered as one of the principal method. However, this approach has major limitations particularly in the developing countries with low-income level of the populations. The second approach includes inclusion of dietary supplements, either by supplementation or through strengthening of the fundamental food items. This process relies on extensive distribution infrastructure and therefore, may not be sustainable for longer period (Hotz and Brown 2004). The third and more advanced approach is known as biofortification, which involves traditional genetic breeding or genetic modification of crops for enhanced accumulation of nutrients (Zhu et al. 2007). In some cases, biofortification has been achieved by the addition of nutrients to fertilizers and found to be successful in enhancing the bioavailability of zinc and selenium (Lyons et al. 2003). The first-generation genetically engineered (GE) crops have shown promising impacts for enhancing agricultural productivity particularly in the developing countries (Farré et al. 2011). The second generation of GE crops, many of which are now under development and trial level, have also been suggested to contribute effectively in reducing micronutrient deficiencies (Yuan et al. 2011).

Transgenic approaches

In transgenic approach, new varieties with desired traits can be developed by introducing new genes or overexpression ofthe genes of interest, or through the inhibition of genes that synthesize inhibitors (Malik and Maqbool 2020). On the other hand, enrichment of micronutrient-associated genes obtained from multiple parents could be difficult to combine into a single genotype by the conventional backcross process. Moreover, the traditional genetic crossing based methods are time and labour intensive (Van Der Straeten et al. 2020). Within this background, transgenic technology represents an alternative and highly efficient biotechnological strategy for the development of multi-nutrient products.

Previously, Goto et al. (1999) have demonstrated successful introduction of the soybean ferritin gene in rice seeds, which showed a threefold increase in Fe content than wild-type seeds. Similarly, Holm et al. (2002) have tried to increase the iron sequestering efficiency in endosperm via in-planta synthesis of microbial phytases. They introduced a PhyA gene with a signal sequence from barley α-amylase into mature embryos of wheat through particle bombardment and a four-fold increase in phytase activity was observed in the transgenic seeds. Subsequent efforts have been given for the improved iron uptake from the rhizosphere and its translocation to further increase iron content in crop plants. It has been shown thatco-expression of Arabidopsisferritin synthase, bean ferritin, along withAspergillus phytase and overexpression of ferritin protein in rice and soybean significantly increase the amount of Fe and Zn, respectively (Wirth et al. 2009). Nicotianamine (NA), the precursor of phytosiderophores, plays a crucial role in transporting micronutrients, such as Fe and Zn to both the vegetative and reproductive organs inplants. Constitutive overexpression of the OsNAS2 gene in rice grains resulted inenhanced accumulation levels of Fe and Zn, respectively (Johnson et al. 2011).Increased iron translocation in rice endosperm was also achieved by the overexpression of another iron transporter gene OsYSL2 (Masuda et al. 2013).Overexpression of OsYSL2 under the control of the OsSUT1(Sucrose transporter 1) promoter sequence showed a significant increase in the accumulation levels Fe, Zn, Mn, and Cu in the seeds of field-grown rice (Masuda et al. 2013). Similarly, overexpression ofArabidopsisAtIRT1, AtNAS1 (NA synthase 1), and bean ferritin in rice caused higher iron and zinc accumulation (Boonyaves et al. 2017). Constitutive overexpression of the Zn-regulated transporter, OsZIP4 in rice resulted in alteration in Zn distribution and this strategy has been suggested for increasing the micronutrient content of other cereal crops (Ishimaru et al. 2007). Recently, a transgenic event named NASFer-274, containing rice OsNAS2 and soybean ferritin SferH-1 genes within a single locus insertion was identified by Trijatmiko and Duenas (2016). In another study, it has been shown that overexpression of Arabidopsis vacuolar Fe transporter (VIT1) in transgenic cassava plantsresulted in increased Fe concentration up to about 70-times comparedto non-transgenic plants (Narayanan et al. 2019). Moreover, this Fe transporter, VIT1has also been found to be involved in Zn and Mn homeostasis (Bashir et al. 2016). Overexpression of AtBOR1 in Arabidopsis showed enhanced boron transport to shoots, resulting in improved shoot growth and yield (Tanaka and Fujiwara 2008).

Nanotechnology

Despite the extensive use of conventional breeding approaches, the reasons behind the lack of improvement in crop yield and plant micronutrient uptake efficiency up to the desired level necessitate new strategies for the improvement of nutritional quality of crops. To overcome the shortcomings of the existing agricultural strategies, nanotechnology has emerged as one of the promising tool with possible potential applications in agriculture (Nile et al. 2020). The increasing development of nanotechnology in the field of medicinal science has spawned the interest inNano-biotechnology for agriculture (Nair et al. 2010). Nanotechnology has received considerable attention for the last couple of years and shown promising impacts in the field of agriculture, food systems, nutraceuticals, diagnostics, therapeutics and pharmaceuticals. Several studies have suggested potential role of nanoparticles to increase the bioavailability of micronutrients in the near future (Arshad et al. 2021; Ndlovu et al. 2020) have shown important role of nanotechnology in enhancing the thermal stability, water solubility and oral bioavailability of nutrients (Ndlovu et al. 2020). The nutritional quality of food depends upon the presence of different micronutrients, such as Fe, Mn, Cu, and Zn. Recent studies have indicated the use of nanotechnology to increase food nutrition quality and fertilizer-micronutrient uptake efficiency (Akhter et al. 2013). Different nanotechnology-based strategies,such as nanoencapsulation, microencapsulation, nanodevices, and nanoparticles have been applied for the improvement of micronutrient use efficiencyin plants (Monreal et al. 2015). Encapsulation of micronutrients not only protects the micronutrients from degradation by pH, light, and oxidants, but also increases the solubility of the less soluble compound and enhances the bioavailability of the micronutrients (Karunaratne et al. 2017). Interestingly, encapsulations of micronutrients in nanoparticles and microcapsules have some potential advantages over the conventional fertilizers. Besides, nanoencapsulated fertilizers have no potential negative ecological impact and their extreme target specificity diminishes the chances of other harmful impacts on the environment. In addition, polymers, such as ethylene-vinylacetate, gelatin, alginate, chitosan, pectin, and starch are used for micronutrient coating, which can further increase the nutrient permeation in the soil (Cui et al. 2010). Different nanoparticles, such as Zn, Fe, Mn, and CuO are used for the controlled release of the micronutrients into the soil (Huo et al. 2014). It has been shown that nanostructures containing Zn at the core with Mn-carbonate coatingincreases Zn uptake efficiency in rice(Yuvaraj and Subramanian 2014). This group has shown that nanoencapsulation of Zn with Mn increases the grain yield of many other crops. Application of ZnO nanoparticles with an average size of 25 nm resulted in an increased Zn use efficiency in corn (Subbaiah et al. 2016).

The highly water-soluble nanoparticles with smaller than 100 nm in size easily penetrate the target plant tissue and interact with other molecules in the plant cell due to their large surface area (Kalra et al. 2020). The foliar application of nano-fertilizers, containing Fe, Mn, Cu, and Zn has been shown to provide considerable agronomic efficiency (Kalra et al. 2020). Nanotechnology has provided the nano-structured components as fertilizer carriers or controlled release of vectors for the improvement of plant nutrient use efficiency. Mesoporous aluminosilicates have been used as carriers ofCuO nanoparticles and have been shown to deliver both macro and micronutrients into the soil (Huo et al. 2014; Pradhan et al. 2013) have shown that nanoparticles containing Mn can increase the activity of the electron transport chain and have the ability to enhance the production of oxygen during photosynthesis. This data indicates that Mn nanoparticles can increase plant growth and development via increasing plant photosynthetic activity. Several other studies have indicated that application of ZnO nanoparticles significantly enhances plant growth (Mahajan et al. 2011; Zhao et al. 2013; Singh et al. 2018). Together, these observations have demonstrated potential applications of nanobiotechnology in enhancing the micronutrient uptake efficiency along with the prevention of contamination of agricultural land and ground water that occur frequently because of excess use of chemical fertilizers.

Advanced genome editing tools

Significant developments in biotechnological approaches during the recent years through hybridization, mutation, and transgenic technologies have paved the way towardsthe development of genetically improved crop varieties (Lusser et al. 2012). In the late 90s, genome manipulation via mutagenesis and transgenic breeding approaches led to theimprovements many traits in crops and helped producing several high-yielding varieties (Ahmad et al. 2022). Advanced biotechnological approaches require comprehensive knowledge of gene sequences, regulatory pathways, cell signaling, enzyme kinetics, and the phenotypic consequences of genetic manipulation, respectively.Modern biotechnological approaches have the potentialto modify or alter different metabolic pathways to increase the content of desirable substances or minerals. However, apart from the advanced techniques, such as the methods of genetic engineering, over expression, RNA interference (RNAi), or genome editing, biotechnological strategies alsoinclude classicalbreeding techniques as well asmarker-assistedselection (MAS). The application of RNAi technology has been suggested to increase the nutritional benefits of some crop species (White and Broadley 2009). The concentration of Fe and Zn has been shown to increase significantly in rice seeds using RNA silencing technologies (Ali et al. 2013). On the other hand, silencing of OsMRP5 in rice seeds reduces the phytic acid content (Li et al. 2012).

The introduction of genome-editing technologies has revolutionized the field of plant sciences.Genomeeditingtechnologies use site-specific endonucleases such as zinc-fingernucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 (CRISPR/Cas9) based systems for targeted mutagenesis, thus immensely helped in elucidating gene function and facilitating improvement of crop plants (Petolino et al. (2010); Noman et al. 2016; Liu et al. 2017). The meganucleases, such as ZFNsand TALENs, have shown great potential for crop improvement via changing the targeted region of any genomes. After the discovery of ZFNs in 1985 and its successful application in plant and animal systems, TALENs were introduced for targeted genetic modifications in plants. Recent studies have demonstrated the implications of meganuclease-based editing systems for genetic manipulations of both model and crop plant genomes, includingArabidopsis,tobacco, rice, wheat, and barley, respectively (Ansari et al. 2020; Jaganathan et al. 2018; Sedeek et al. 2019). TheTALENs mainly contain an engineered DNA-binding domain of a TALE fused to a nonspecific FokI cleavage domain. TALENs were first successfully introduced in rice for trait improvement (Li et al. 2012). Subsequent studieshave reported a region in the promoter of barley phytase gene,HvPAPhy, containing a group of regulatory motifs (Wendt et al. 2013). These regulatory motifs were targeted by TALENS for the manipulation of the phytase gene. Previously, Shan et al. (2015) reported the application of specially designed TALENs for the mutation in OsBADH2 gene in rice, leading to increased aroma quality. Haun et al. (2014) reported the development of high oleic acid containing soybean varieties through targeted mutagenesis of fatty acid desaturase genes, such as FAD2-1 A and FAD2-1B using TALENs. Clasen et al. (2016) reported the knockout mutation of the vacuolar invertase gene (VInv) in potato by using TALENs, leading to improvement of quality desirable required for processing of potato.However, ZFNs and TALENs are both cost and time intensive, particularlythe designing of these proteins is time-consuming as compared to other advanced base editing systems (Shiva Krishna and Suma 2019). In addition, high rates of off-target effects are one of the major issues associated with these systems and has been shown to cause significant level of cytotoxicity (Radecke et al. 2010).

Programmable and site-specific CRISPR-Cas-based gene editing mechanism aremore or less similar to ZFNs and TALENs, butendonuclease is guided to the target site by an RNA sequence (sgRNA). The guide RNA recognizes the target sequence in the genome and CRISPR-associated endonuclease (Cas) then cleaves the target site (Sun et al. 2020). CRISPR-Cas based system can edit any sequence in the genome and hence this system emerged as a more robust, efficient, and cost-effective gene editing technology than ZFN- and TALEN-based genome editing systems (Zhang 2020).The application of base editing and prime editing further improved the CRISPR-based editing systems by minimizing off-target effects. In rice, OsZIP9, a member of Zn-IRT-related proteinhas been knocked out by the CRISPR/Cas9 system. The knockout mutants showed decreased Zn contentin roots and shoots and thus indicated the function of OsZIP9 as a flux carrier of Zn in rice (Huang et al. 2020). The CRISPR/Cas9 system has beensuccessfully used in crop plants to inhibitthe excess accumulation of toxic micronutrients, such asCd. For example, theOsNramp5 knockout mutant lines in rice showed significant enhancement in the expression of Fe/Cd transporter genes, such as OsIRT1, andOsIRT2under Fe deficiency (Takahashi et al. 2014). In addition,the osnramp5 mutants in rice showed decreased Cd accumulation in seedlings along with improved growth and developmental response (Tang et al. 2017). Editing of genes associated with micronutrient accumulation through genome editing techniqueshas great advantages over the traditional breeding tools because of their high precision in fine-tuning target gene expression. CRISPR-based systems can also effectively modify the regulatory elements of target genes associated with micronutrient accumulation. Several candidate genes involved in Fe and Zn homeostasis, including OsNAS2, OsYSL2, OsIRT2, OsDMAS1, and OsYSL15 were identified and found to play crucial role in iron uptake from soil. Therefore, these transporters represent some of the potential targets for genome editing (Anuradha et al. 2012). Recent studies have shown CRISPR/Cas9 mediated targeting of four yield-related genes in rice, such as Gn1a, DEP1, GS3, and IPA1 for the improvements of yield traits (Li et al. 2016). Furthermore, the development of hybrids via base editingalso offers an important method to improve desired traits withina very short time period and may lead to several-fold increase in crop yield and grain nutrient value. CRISPR-Cas9 system has emerged as a new generation breeding tool in the development of climate resilient crops, induction of self-compatibility, enhancement of nutrient content in crop plants, and therefore,will probably help to meet the global challenge of food security in the long run. One important concern of CRISPR-Cas9 based genome editing tool is the large genome size of some crop plants, which contain multiple repetitive sequences and thus may create many off-target cuts. However, in recent times,various techniques have been developed for the identification of off-target mutations,which further strengthen thistechnology for the targeted modification of genes associated with micronutrient assimilation.

Conclusion and future perspectives

Micronutrients are essential formaintaining plant growth and development via regulating cellular metabolism and protein synthesis, as they arethe key structural components of many co-enzymes and macromolecules, such as amino acids. During the last decade a comprehensive knowledge has been gained from the different published literatures (Fig. 3), which clearly demonstrated the function of microelements in the maintenance of plant growth and development. Between 2011 and 2013, initially, few articles were published, describing the biofortification of iodine (I) for the improvement of plant nutrient use efficiency and defence signalling (Tonacchera et al. 2013). During the subsequent years (2013 onwards), significant number of review papers have elucidated the crucial role of Fe in seed and leaf development in plants. Meanwhile, various studies began to publish, focusing on the areas of genetic engineering and biotechnological approaches for nutritional (Fe and Zn) enhancement of rice endosperm and other crop plants (Bhullar and Gruissem 2013; Mari et al. 2020). Subsequent studies have indicated important role of Zn and Ni in the development of plant defense mechanisms (Polacco et al. 2013; Assunção et al. 2013). Moreover, during the last decade, several review papers have mentioned plant responses to micronutrient (Fe, Zn, Mn) deficiency (Assuncao et al. 2013; Schmidt et al. 2016; Kobayashi et al. 2019). In recent years (2020–2022), most of the research and review works have discussed the function of transporters and their molecular regulation for the enhancement of micronutrient use efficiency in plants. Furthermore, microelements are involved in an array of cellular biosynthetic and other metabolic processes. Micronutrients also play crucial role in cognitive and physical development, reproductive well-being, and immune responses in humans and animals. As humans and animals are directly dependent on plants for food, deficiency in nutrient content in plants directly causes several health issues in humans and animals. Micronutrient deficiency exerts substantial human health issues globally, creating serious health and nutritional consequences in an estimated two billion human beings. On the other hand, climate change and environmental pollution directly or indirectly influence grain mineral concentration. Therefore, comprehensive knowledge on plant genetics and molecular mechanisms that regulate macro and micronutrient uptake will help to improve grain’s nutrient quality. However, instead of any specific technology, a combinational approach, involving both traditional and genetic manipulation techniques will be beneficial to address the challenge of micronutrient deficiency. Conventional breeding approaches based on QTLs and MAS, are although time and labour intensive process, have shown considerable level of success in the improvements of various crops (Kumawat et al. 2020).On the other hand, certain advanced technologies in agricultural practices have been applied for enhancement of grain quality. Recently, in-silico approaches have been employed in the identification of genes involved in regulating micronutrient uptake in plants (Fig. 2). The identification of promoter regions of differentially expressed miRNAs or the binding sites of TFs, which are responsive to micronutrient uptake, have been identified as the key targets for manipulation through advanced genome editing biotechnological tools, including TALEN and CRISPR-Cas9, respectively (Fig. 2).

Fig. 3.

Analysis of the trends in the literature published in the last decade mentioning plant micronutrient use efficiency. A graphical representation showing the number of research and review articles published during 2011 to 2022. The number of publications during the last decade was checked using PubMed database. Under the search criteria of ‘Plant micronutrient use efficiency,’ significant number of review and research papers published between 2011 and 2022 were found. Initially, between 2011 and 2013, only few papers were found to be published, describing the biofortification of iodine (I) for the improvement of plant nutrient use efficiency and defense signaling. During the subsequent, the number of publications, both research and review articles increased significantly, showcasing pivotal role of micronutrients in regulation of plant development and the biotechnological strategies for enhancement of micronutrient use efficiency in plants

The United Nations Sustainable Development Goals have announced for the end of the global hunger and aiming towards reducing the various forms of malnutrition by the year 2030 (United Nations. Sustainable Development Goal 2: end hunger, achieve food security and improved nutrition and promote sustainable agriculture. United Nations Sustainable Development Knowledge). As indicated in several previous reports, resolving the issues of micronutrient deficiency will still remain one of the major challenges for the coming years (Mayer et al., 2008; McGloughlin 2010). Alongside micronutrient deficiency or hidden hunger, inadequate dietary intake of necessary minerals and vitamins has also imparted severe effects on human health. Currently, approximately two billion population worldwide is affected by micronutrient deficiency or hidden hunger (White and Broadley 2011; Ritchie et al. 2018; Ibeanu et al. 2020) and the impacts are more prominent in the African continent and South Asian countries. In these regions, the infants and particularly women at the reproductive stage are mostly affected by malnourishment, lacking sufficient dietary minerals and vitamins, leading to severe growth problems and various birth related issues. Within this context, genetic modification of plants has been considered as one of the crucial strategies for the improvements of nutritional elements along with the enhancement of bioavailability of macro and microelements in the cereal crops. Genetic modifications may be achieved either through conventional breeding or genetic engineering approaches for the enrichment of nutrients in staple crops and enhancement of nutrient use efficiency. During the recent years, agricultural biotechnology has emerged as one of the important tools for the enhancement of levels of micronutrients, vitamin A, folate, Zn and Fe in the staple crops, including rice in the resource-poor countries (Zhu et al. 2007). As part of an important tool in agricultural biotechnology, biofortification serves as an important approach for the enrichment of micronutrients to food crops, while dietary diversification and mineral supplementation facilitates to relieve micronutrient deficiency. Recent studies have elucidated the potential role of biofortification tool through agricultural biotechnologies alongside the combinatorial approaches of dietary diversification and mineral supplementation to mitigate micronutrient deficiency and improve the global human health (Gartland and Gartland 2018; Straeten et al. 2020). Biofortification through agricultural biotechnology also relies on the precise genome editing approaches, including the CRISPR-Cas9 and other genome editing strategies and also on the genetic manipulation of regulatory elements, like microRNAs. Besides genome editing, during the recent years, emphasis has been given on the other biotechnological approaches, such as the microRNA mediated regulation of nutrient transporters and metabolic enzymes under the nutrient stress conditions in cereal crops (Islam et al. 2022). Along with the traditional and marker-assisted plant breeding approaches, biofortification of crops through agricultural biotechnology using less-expensive, rapid and more potential genome editing strategies will provide important tool in the long run to overcome the challenge of global micronutrient malnutrition. However, the time and cost-intensive regulatory constraints for obtaining marketing approval of the products from governments, the concern with the GMO crops in connection with the distribution of the nutrient enriched biofortified crops to the rural areas in developing countries still represent some major hurdle and will remain a principal challenge in future (Pérez-Massot et al. 2013; Potrykus 2010; Sperotto et al. 2012). On the other hand, scientists from all over the globe also have a key role in transferring the knowledge from the laboratory and field to the regulatory bodies and society in a more convincing way to unveil both the advantages and disadvantages of the crops generated through biotechnological approaches. Considering the recent COVID-19 mediated global crisis and the very prominent sign and impacts of climate change, the process of knowledge transfer has now become more crucial in the context of the issues of how biotechnology mediated crops would be addressed in the future.

Author contributions

SR and SB conceptualized the idea. SR and SB wrote the manuscript. SB and PR prepared the figures. SR, SB, PR, and SN edited the manuscript. SR critically revised the manuscript draft to the final version to be published. All authors reviewed and approved the final version of the manuscript.

Declarations

Conflict of interest

No potential competing interest is reported by the authors.