**3. Progress in improving nutritional quality**

allow further manipulation of the nutrient loading pathway without affecting other traits of importance [31, 32]. Thereof, the selected omics technological platforms would bring about data outputs that would allow the establishment of a good balance in the expression of selected traits of interest in desired grain compartments [33]. The integration of these technologies would allow researchers to identify novel genes or pathways that could be activated to improve the bioavailability of desired nutritional components in wheat. This chapter aims to highlight the progress and challenges encountered in attempts to improve nutritional quality in wheat in order to recommend strategies that could be deployed to improve nutritional quality in a more sustainable and efficient way. The most important research question that needs to be addressed is, what is the source or origin of the total grain nutrient content of minerals or phenolic acids found in different grain compartments? Thus, there is still a need to conduct a quantitative assessment of the total mineral nutrient use efficiency and the type of mineral used for plant

Wheat, grown in many parts of the world, is a major contributor to food security in that it is a staple food in other countries [1]. It has three main grain compartments such as the bran, endosperm and the germ. The wheat grain as a whole houses a series of nutritional health beneficial components ranging from macronutrients, micronutrients, vitamins, phenolic compounds and other components at different levels across various grain compartments [2]. The wheat grain is also a major contributor to the daily dietary intake required by individuals due to its regular consumption in various forms. Thus, regular consumption of essential nutrients at adequate levels could largely contribute to the reduction of nutrient deficiency-related ailments such as anaemia, growth and development problems, cardiovascular diseases, cancer,

Intriguingly, the endosperm region is the most edible part of the grain reported to contain less contents of Fe and Zn than the outer layers that are removed upon milling [18, 34]. Several efforts to establish the biofortification of wheat have been undertaken and some major challenges have been experienced. Little or no progress has been made to characterize the key biological process involved in the accumulation and bioavailability enhancement of Fe, Zn,

Wheat has a complex genome and the complete genome sequence is not available yet. This makes it challenging to identify and understand the function of many genes in wheat, thereby making it difficult to characterize and manipulate complex traits of interest for the development of improved varieties. Further characterization of some traits is still needed for a continued contribution to better understand various gene networks/pathways and their role within the wheat genome to allow rapid development of improved cultivars with desirable traits of interest for a continued contribution to food and nutrition security. There are various wheat genetic resources ranging from landraces to wild relatives that may carry various genes of interest due to their genetic diversity [1, 35, 36]. Genetic resources have been utilized for crop improvement efforts in cases where information regarding a complex trait is not readily known, the informa-

tion may then be inferred from closely related species with known biology [1].

metabolism and seed production.

348 Wheat Improvement, Management and Utilization

diabetes, neurological disorders, etc. [7].

vitamins and phenolic acids in this grain compartment [14].

**2. Wheat**

The improvement of nutritional quality entails a series of processes to ensure that the nutrients are bioavailable upon consumption. The major process requires a genotypic and phenotypic characterization of key biological processes or pathways that are involved in the assimilation, accumulation, biosynthesis, translocation and remobilization of desired nutritional quality components such as Fe, Zn, vitamins and phenolic acids in the wheat grain [12, 15, 37–39]. The ultimate process involves the application of biofortification, which is the most sustainable approach that can reach the nutritional requirements of the global community in a cost-effective manner. However, the application of biofortification requires rudimentary information regarding the crop's genetic and phenotypic profile across different environments. Substantial progress has been made in attempts to improve nutritional quality in wheat. This includes the deployment of several strategies that involve the application of conventional, technological and transgenic approaches [14, 40].

Conventional-based approaches involve the use of basic genetic and agronomic practices, such as agronomic biofortification, soil+foliar application and genetic biofortification, which involves germplasm screening to reveal the genetic variation for grain Fe and Zn levels across different wheat genotypes grown in different environments [21]. Progress has been made to establish genetic variation of Fe and Zn across various wheat species. Along the process, an important quantitative trait locus (QTL) *Gpc-B1* from wild emmer wheat (*Triticum turgidum* ssp. *dicoccoides*) was discovered and mapped on chromosome arm 6BS [41]. The gene of this locus was then cloned and effectively improved Zn, Fe and protein concentrations by 12%, 18% and 38%, respectively [19]. The *Xuhw89* marker is linked to the *Gpc-B1* locus with a 0.1 cM genetic distance and can be used to identify and select lines with improved levels of selected micronutrients in the wheat grain [42]. In addition, several efforts have also been made to establish the genetic variation in the levels of phenolic compounds in some wheat species.

Technological-based approaches involve the application of advanced high-throughput analytical technologies such as ribonucleic acid sequencing (RNAseq), ribonucleic acid interference (RNAi), genomics, transcriptomics and metabolomics to discover and characterize candidate genes that could be used to improve nutritional quality. This may also include genome editing-based approaches such as the CRISPR Cas9 approach, which has recently been used in wheat [20]. Transgenic-based approaches mainly involve the application of genetic modification to improve nutrient accumulation in the wheat grain. Some minimal progress has been made with the application of transgenic approaches in attempts to improve nutritional quality [43, 44].

Several applications have been deployed to improve nutritional quality in wheat; some applications were successful but not sustainable and others were not successful [12, 13, 28]. Technological applications have also been deployed for wheat improvement. This includes success in increasing the bioavailability of Fe and Zn and decreasing the antinutrients such as phytic acid and polyphenols, which inhibit Fe absorption thereby reducing Fe bioavailability. However, a series of strategies to improve the bioavailability of micronutrients and phenolic acids have been deployed, this includes agronomic biofortification and the use of nutritional enhancers [27, 28, 38]. Micronutrients and phenolic acids have also been reported to be present at high concentrations in the outer layers of the seed and in the wheat germ region than in the endosperm region [18, 34].

Nonetheless, there are challenges with the biofortification of wheat with other nutritional components. This is mainly due to an incomplete understanding of pathways involved in the translocation of desirable nutritional components into desired wheat grain compartments such as the endosperm. In addition, the bioavailability of micronutrients such as Fe is reduced due to its interaction with other anti-nutritional components such as phytic acid or the food matrix, which constitutes other nutritional or anti-nutritional wheat grain components [43, 45, 46]. Moreover, the wheat endosperm region was also reported to lack transporters that are essential for the translocation Fe into the endosperm region [14, 47]. little or no research has been conducted to manipulate the transporter proteins to translocate more Fe into the wheat grain. Little or no studies were conducted on the translocation of phenolic acids into the wheat endosperm, and there is less information regarding the translocation or transporters involved in the translocation of phenolic compound and vitamins into the endosperm region.

A number of attempts to address the above-mentioned challenges were undertaken through the application of various conventional, technological and transgenic approaches. Much progress has been made in attempts to understand key processes involved in the assimilation, translocation and biosynthesis of micronutrients into the wheat grain [12–15, 48]. However, there is still a great need to utilize selected omics technologies to further improve our understanding on processes involved in optimising the accumulation of essential nutrients in the wheat grain.

#### **3.1. Establishing genetic variation**

The establishment of genetic variation entails screening various wheat genotypes grown across different environments for their levels of total Fe, Zn, vitamins and phenolic acids found in the wheat grain with the aid of analytical instruments [49–51]. Most or all studies on genetic variation in grain Fe and Zn concentration reported on the total grain Fe and Zn concentration obtained in wholemeal flour. Little or no reports are available on establishing the genetic variation in grain Fe and Zn concentration in white flour. However, the Agricultural Research Council-Small Grain Institute of South Africa has reported some preliminary data on the levels of Fe and Zn found in white flour among some modern commercial wheat genotypes, which showed some degree of genetic variation at a local conference in 2016 (unpublished data).

Velu et al. [28] reported substantial progress made on screening more than 7800 wheat genotypes for their variation in Zn concentration in bread wheat, durum wheat, wheat landraces and their wild relatives from several studies conducted since 1983 until 2012. The studies from the paper revealed some genotypes that had the highest grain Zn concentration reaching as far as 142 mg/kg, whereas other wheat genotypes especially the improved adapted wheat genotypes showed little variation in grain Zn [28]. Amiri et al. [52] also reported the genetic variation for grain's protein, Fe and Zn concentration among 80 irrigated bread wheat genotypes, which showed some level of genetic variation. Gorafi et al. [53] also reported the assessment of genetic variation in grain Fe and Zn concentrations in more than 40 synthetic hexaploid wheat lines and conducted further development of the wheat lines for use as genetic resources. Thus, various wheat genotypes showed a significant genetic variation in wheat grain Fe and Zn content. Consequently, wheat genotypes that contain the highest levels of Fe and Zn could be selected as donors to improve the levels of Fe and Zn in recurrent parents who have lower levels of Fe and Zn. However, it is imperative to ensure that important traits, such as grain yield, protein content, disease resistance and other agronomic traits, are not compromised upon the development of varieties with improved nutritional quality.

Genetic variation was reported in phenolic acid content of various wheat genotypes [54–56]. Thus, some progress has been made to selectively breed for genotypes with the highest phenolic acid content. However, more studies are needed to further confirm the genetic variation that exists in phenolic acids among different wheat genotypes through germplasm screening of other wheat genotypes including wild relatives and landraces.

Little or no research has been reported on the establishment of genetic variation on the concentration of vitamins, manganese, magnesium, copper, potassium, as well as concentrations of other anti-nutritional components found in the wheat grain. Nonetheless, [49, 50, 57] provided a report on the levels of tocol (vitamin E) content found in various wheat genotypes. However, more studies are needed in this field.

The establishment of genetic variation in minerals has led to the improvement of several wheat germplasms. The selected genotypes were used to improve the levels of Zn by more than twofold in other instances [28]. However, there are some drawbacks with conventional breeding, in that it may take several years to develop a new variety with improved nutritional quality. In addition, only the total grain Fe and Zn can be increased. Therefore, breeders have no control on improving the levels of selected nutrients into desired grain compartments.

### **3.2. Grain nutrient content**

ent at high concentrations in the outer layers of the seed and in the wheat germ region than in

Nonetheless, there are challenges with the biofortification of wheat with other nutritional components. This is mainly due to an incomplete understanding of pathways involved in the translocation of desirable nutritional components into desired wheat grain compartments such as the endosperm. In addition, the bioavailability of micronutrients such as Fe is reduced due to its interaction with other anti-nutritional components such as phytic acid or the food matrix, which constitutes other nutritional or anti-nutritional wheat grain components [43, 45, 46]. Moreover, the wheat endosperm region was also reported to lack transporters that are essential for the translocation Fe into the endosperm region [14, 47]. little or no research has been conducted to manipulate the transporter proteins to translocate more Fe into the wheat grain. Little or no studies were conducted on the translocation of phenolic acids into the wheat endosperm, and there is less information regarding the translocation or transporters involved in the translocation of phenolic compound and vitamins into the endosperm region. A number of attempts to address the above-mentioned challenges were undertaken through the application of various conventional, technological and transgenic approaches. Much progress has been made in attempts to understand key processes involved in the assimilation, translocation and biosynthesis of micronutrients into the wheat grain [12–15, 48]. However, there is still a great need to utilize selected omics technologies to further improve our understanding on processes involved in optimising the accumulation of essential nutrients in the

The establishment of genetic variation entails screening various wheat genotypes grown across different environments for their levels of total Fe, Zn, vitamins and phenolic acids found in the wheat grain with the aid of analytical instruments [49–51]. Most or all studies on genetic variation in grain Fe and Zn concentration reported on the total grain Fe and Zn concentration obtained in wholemeal flour. Little or no reports are available on establishing the genetic variation in grain Fe and Zn concentration in white flour. However, the Agricultural Research Council-Small Grain Institute of South Africa has reported some preliminary data on the levels of Fe and Zn found in white flour among some modern commercial wheat genotypes, which showed some degree of genetic variation at a local conference in 2016 (unpub-

Velu et al. [28] reported substantial progress made on screening more than 7800 wheat genotypes for their variation in Zn concentration in bread wheat, durum wheat, wheat landraces and their wild relatives from several studies conducted since 1983 until 2012. The studies from the paper revealed some genotypes that had the highest grain Zn concentration reaching as far as 142 mg/kg, whereas other wheat genotypes especially the improved adapted wheat genotypes showed little variation in grain Zn [28]. Amiri et al. [52] also reported the genetic variation for grain's protein, Fe and Zn concentration among 80 irrigated bread wheat genotypes, which showed some level of genetic variation. Gorafi et al. [53] also reported the assessment of genetic variation in grain Fe and Zn concentrations in more than 40 syn-

the endosperm region [18, 34].

350 Wheat Improvement, Management and Utilization

wheat grain.

lished data).

**3.1. Establishing genetic variation**

Wheat grain houses a number of nutritional components ranging from macronutrients, micronutrients, vitamins, amino acids, arabinoxylans and various other nutritional components [2]. These components vary in quantity due to the manner in which they are incorporated into different grain compartments upon seed formation. Thus, increasing the quantity of selected nutritional component might result in a decrease in other constituents [21, 43]. Hence, it would be ideal to optimize the production of desirable nutrients in a manner that could result in the reduction of non-targeted wheat grain components. However, this would be a major challenge in that some or most traits in certain organisms are quantitative and the expression of a selected trait could depend on the expression of more than one gene, thereby resulting in minimal expression or production of a desired nutritional component.

Starch, protein and cell wall polysaccharides (dietary fibre) are the major grain nutritional components that account for about 90% of the dry weight and minerals, vitamins, lipids, phenolic compounds and terpenoids are among the minor grain nutritional constituents found in wheat. A major component of the endosperm comprises about 80% of starch and about 10% of other constituents, including minerals and some phytochemicals, which are mostly concentrated in the wheat bran area [58].

The levels of minerals in several wheat varieties particularly Fe and Zn have been reported to be declined over more than five decades due to their dilution with starch [2]. Nonetheless, substantial progress has been made in improving the total grain nutrient content with micronutrients such as Fe and Zn. The second HarvestPlus Yield Trial has managed to improve the levels of 50 wheat lines through biofortification with a total grain Zn content, which was 75–150% more than the control lines used for the trial [28, 59]. Hidalgo and Brandolini [60] reported that the wheat bran region of some einkorn accessions and some bread wheat genotype had the highest levels of total tocols, including α–tocopherol and β-tocopherol, in a study that screened the distribution of tocols across different grain compartments.

Agronomic biofortification, a traditional biofortification approach, which involves direct micronutrient uptake from the soil that gets remobilised into the grain, has been applied in wheat to improve the levels of Fe and Zn. Much progress has been made in the application of this strategy for the biofortification of wheat with grain Zn. This was done through the application of Zn fertilizers using the soil and foliar application method, which can result in about threefold increase in the total grain Zn concentration [21]. Several studies that involved the use of radioactive Fe and Zn were carried out to evaluate ways to gain better understanding of the remobilisation of selected minerals [19, 32, 61]. The studies largely contributed to depicting the manner in which micronutrients are translocated into seeds from various tissues. Feil et al. [62] reported that environmental conditions, particularly soil composition, largely influence the total micronutrient concentration of wheat grain. Thus, agronomic biofortification can facilitate nutrient uptake and ultimately improve the total grain Zn content. However, this process is mainly dependent on the availability of minerals in the soil or through provision from the fertilizer, thereby making it an unsustainable approach to utilize in improving nutritional quality.

#### *3.2.1. Nutrient translocation into grain*

There are genes that contribute to the translocation of minerals, mainly Fe and Zn into the wheat grain. Nutrient uptake and translocation or remobilization are complex processes that are involved in seed nutrient loading to make up the total grain nutrient content [12, 15, 21]. The two major processes involved in nutrient uptake and translocation and/or remobilization are mainly dedicated for plant metabolism and seed production. In the case of plant metabolism, nutrients would be taken up, translocated or remobilized to specific tissues in response to growth and developmental requirements, including mineral deficiencies. Whereas in the case of seed production, the source of the total nutrient content found in the seed remains unknown because nutrient loading in the seed has been attributed to multiple processes including senescence and direct translocation with the aid of transporters [19, 32]. The process of moving micronutrients from the soil into the seeds is a complex process, which still requires further characterization. Waters and Sankaran [15] provided a review uncovering several processes involved in the improvement of seed mineral biofortification on various species, including wheat, and made a recommendation that the simultaneous enhancement mineral uptake from roots to shoots and ultimately remobilization into seeds would result in successful seed mineral biofortification.

Nutrient remobilization through senescence was reported to be more efficient in cases where the plant carries a Gpc-B1 locus derived from *T. dicoccoides* [21]. Wherein, Distelfeld et al. [42] showed that recombinant substitution lines (RSLs) carrying the *dicoccoides* Gpb-B1 allele had 12%, 18% and 38% more Zn, Fe and grain protein content (GPC), respectively, than (RSLs) carrying a Gpc-B1 locus acquired from durum wheat. Thus, there is a great need to distinguish whether the nutrients that are accumulated in the grain are excess nutrients that were committed for plant development in the leaves, which are translocated into the wheat grain upon senescence or whether they are accumulated and translocated into the wheat grain during different growth developmental stages. Consequently, there is a great need to trace the origin of nutrients found in different grain compartments.

#### *3.2.2. Transgenic approaches*

The levels of minerals in several wheat varieties particularly Fe and Zn have been reported to be declined over more than five decades due to their dilution with starch [2]. Nonetheless, substantial progress has been made in improving the total grain nutrient content with micronutrients such as Fe and Zn. The second HarvestPlus Yield Trial has managed to improve the levels of 50 wheat lines through biofortification with a total grain Zn content, which was 75–150% more than the control lines used for the trial [28, 59]. Hidalgo and Brandolini [60] reported that the wheat bran region of some einkorn accessions and some bread wheat genotype had the highest levels of total tocols, including α–tocopherol and β-tocopherol, in a study

Agronomic biofortification, a traditional biofortification approach, which involves direct micronutrient uptake from the soil that gets remobilised into the grain, has been applied in wheat to improve the levels of Fe and Zn. Much progress has been made in the application of this strategy for the biofortification of wheat with grain Zn. This was done through the application of Zn fertilizers using the soil and foliar application method, which can result in about threefold increase in the total grain Zn concentration [21]. Several studies that involved the use of radioactive Fe and Zn were carried out to evaluate ways to gain better understanding of the remobilisation of selected minerals [19, 32, 61]. The studies largely contributed to depicting the manner in which micronutrients are translocated into seeds from various tissues. Feil et al. [62] reported that environmental conditions, particularly soil composition, largely influence the total micronutrient concentration of wheat grain. Thus, agronomic biofortification can facilitate nutrient uptake and ultimately improve the total grain Zn content. However, this process is mainly dependent on the availability of minerals in the soil or through provision from the fertilizer, thereby making it an unsustainable approach to utilize in improving

There are genes that contribute to the translocation of minerals, mainly Fe and Zn into the wheat grain. Nutrient uptake and translocation or remobilization are complex processes that are involved in seed nutrient loading to make up the total grain nutrient content [12, 15, 21]. The two major processes involved in nutrient uptake and translocation and/or remobilization are mainly dedicated for plant metabolism and seed production. In the case of plant metabolism, nutrients would be taken up, translocated or remobilized to specific tissues in response to growth and developmental requirements, including mineral deficiencies. Whereas in the case of seed production, the source of the total nutrient content found in the seed remains unknown because nutrient loading in the seed has been attributed to multiple processes including senescence and direct translocation with the aid of transporters [19, 32]. The process of moving micronutrients from the soil into the seeds is a complex process, which still requires further characterization. Waters and Sankaran [15] provided a review uncovering several processes involved in the improvement of seed mineral biofortification on various species, including wheat, and made a recommendation that the simultaneous enhancement mineral uptake from roots to shoots and ultimately remobilization into seeds would result in

that screened the distribution of tocols across different grain compartments.

nutritional quality.

*3.2.1. Nutrient translocation into grain*

352 Wheat Improvement, Management and Utilization

successful seed mineral biofortification.

A transgenic approach that could enhance the Fe concentration in edible plant part is the overexpression of ferritin, an Fe-rich soybean (*Phaseolus vulgaris*) storage protein [63, 64], which completely degrades phytate in seeds. Ferritin is considered a more bioavailable storage form and is abundant in the endosperm amyloplasts, the widely consumed grain compartment [65]. Ferritin genes of soybean were introduced and used to produce transgenic rice lines, and the concentrations of Fe were doubled with the highest Fe level in the transgenic lines [63]. Recombinant soybean ferritin gene also increased seed Fe concentration in rice, under the control of an endosperm-specific promoter [66, 67].

However, ferritin overexpression possesses a disadvantage in transgenic crops as the accumulation of Fe might depend greatly on the soil composition, for example, transgenic tobacco (*Nicotiana tabacum*) continuously overexpresses ferritin under a 35S-GUS promoter [68] and Fe deficiency was widespread in the crop. Metals, such as cadmium, lead and nickel, which are toxic for human health, were found rich in ferritin-overexpressing tobacco plants, when grown in one of the tested soil [68]. Consequently, Fe accumulation within ferritin results in an iron deficiency in these transgenic tobacco plants [68]. Iron deficiency expresses ferrous Fe root transporters, which also uptake cadmium, thereby promoting cadmium accumulation in plants [69–74].

#### **3.3. Candidate genes for nutritional quality enhancement**

Nutrient biosynthesis and accumulation in the seed involves multiple complex processes. Phenolic acids are mainly synthesized from phenylalanine, a major precursor molecule for the phenlypropanoid biosynthetic pathway [39, 75, 76]. The biosynthesis of phenolic acids is mainly governed by several genes, which encode enzymes to carry out biochemical reactions involved in the production of selected phenolic acids. However, little or no information is known on the process that is involved in the loading of specific phenolic acids into different grain compartments. Micronutrient accumulation in the wheat grain is mainly dependent on the availability of soil mineral nutrients, which are taken up from the roots and then translocated to different plant compartments. In this process, a series of genes and active transport protein families are activated to facilitate in the nutrient translocation and remobilization process. The total quantity of micronutrients found in different grain compartments depend on environmental circumstances and the growth stage in which micronutrients are taken up, translocated or remobilized from different plant tissues. Thus, it would depend on the nutrient soil status and the stage at which the selected nutrients are taken up. Nonetheless, there is little or no research on the characterization of the origin and starting concentration of the nutritional component attributed to specific concentrations obtained in specific grain compartments.

#### *3.3.1. Genes involved in micronutrient accumulation*

Waters and Sankaran [15] reported genes implicated in the uptake of Fe mainly. No gene(s) that are involved in Fe uptake have been reported for wheat. Thus, there is still a need to characterize and identify genes involved in the uptake of Fe from soil to the seeds in wheat. Furthermore, [70, 77, 78] provided a comprehensive overview of genes and pathways involved mainly in Fe uptake from roots to other plant compartments. Waters et al. [61] conducted a more comprehensive investigation on the role of the *NAM-B1* gene, which affects Fe and Zn in wheat.

*Gpc-B1* locus from *Triticum dicoccoides* was mapped and found to enhance Zn and Fe concentrations and encoded a NAC transcription factor that was found responsible to accelerate senescence. Senescence, the programmed degradation of cell constituents makes nutrients available for remobilization from leaves to developing seeds [19, 42]. Kohl et al. [79] reported that some NAC transcription factors were upregulated in the glumes at 14 days after anthesis and were obviously associated with developmental senescence. During senescence, proteases are rapidly activated to degrade leaf proteins into amino acids [80]. Serine proteases are the most important family of proteases participating in nitrogen remobilization (NR) during grain filling, acting as major regulators and executors in wheat and barley [81].

In wheat and barley, the specific NAC and WRKY transcription factors, in combination with hormones (abscisic acid and jasmonic acid), have been shown to be involved in the regulation of transition between early grain filling and developmental senescence [79, 82, 83]. Zhao et al. [84] identified a novel NAC1-type transcription factor, TaNAC-S, in wheat, with gene expression located primarily in the leaf/sheath tissues. Overexpression of TaNAC-S in transgenic wheat plants resulted in delayed leaf senescence, which led not only to increased GPC but also to increased grain yields; thus, this result further verified the improved NR from vegetative organs to growing grain in transgenic lines [84].

#### *3.3.2. Genes involved in phenolic acid accumulation*

Very little research has been conducted on the accumulation of phenolic acids. Ma et al. [39] reported five key enzymes, namely phenylalanine ammonia lyase (PAL), coumaric acid 3-hydrolase (C3 H), cinnamic acid 4-hydrolase (C<sup>4</sup> H), 4-coumarate CoA ligase (4CL) and caffeic acid/5-hydroxyferulic acid O-methlytransferase (COMT), which are essential for the biosynthesis of phenolic acids. Ma et al. [39] also characterized gene expression patterns of nine candidate genes associated with phenolic acid biosynthesis during early and late grain filling stages, the most crucial growth stage in polyphenol accumulation [85, 86]. The study revealed that seven genes (*TaPAL1*, *TaPAL2, Ta4CL1, Ta4CL2, TaCOMT1, TaCOMT2* and *TaC3H2*) are highly expressed during the early stages of grain development among white, red and purple wheat. However, *TaC3H1* was the single gene that was expressed only during the later stage of grain development. Finally, five genes (*TaC4H, TaPAL1, TaPAL2, Ta4CL2 and TaCOMT1*) showed higher expressions in both early and later grain developmental stages [39]. Hence, there is still a need to conduct studies to further characterize the process of phenolic acid accumulation in seeds.

on environmental circumstances and the growth stage in which micronutrients are taken up, translocated or remobilized from different plant tissues. Thus, it would depend on the nutrient soil status and the stage at which the selected nutrients are taken up. Nonetheless, there is little or no research on the characterization of the origin and starting concentration of the nutritional component attributed to specific concentrations obtained in specific grain compartments.

Waters and Sankaran [15] reported genes implicated in the uptake of Fe mainly. No gene(s) that are involved in Fe uptake have been reported for wheat. Thus, there is still a need to characterize and identify genes involved in the uptake of Fe from soil to the seeds in wheat. Furthermore, [70, 77, 78] provided a comprehensive overview of genes and pathways involved mainly in Fe uptake from roots to other plant compartments. Waters et al. [61] conducted a more comprehensive investigation on the role of the *NAM-B1* gene, which affects Fe and Zn in wheat.

*Gpc-B1* locus from *Triticum dicoccoides* was mapped and found to enhance Zn and Fe concentrations and encoded a NAC transcription factor that was found responsible to accelerate senescence. Senescence, the programmed degradation of cell constituents makes nutrients available for remobilization from leaves to developing seeds [19, 42]. Kohl et al. [79] reported that some NAC transcription factors were upregulated in the glumes at 14 days after anthesis and were obviously associated with developmental senescence. During senescence, proteases are rapidly activated to degrade leaf proteins into amino acids [80]. Serine proteases are the most important family of proteases participating in nitrogen remobilization (NR) during

In wheat and barley, the specific NAC and WRKY transcription factors, in combination with hormones (abscisic acid and jasmonic acid), have been shown to be involved in the regulation of transition between early grain filling and developmental senescence [79, 82, 83]. Zhao et al. [84] identified a novel NAC1-type transcription factor, TaNAC-S, in wheat, with gene expression located primarily in the leaf/sheath tissues. Overexpression of TaNAC-S in transgenic wheat plants resulted in delayed leaf senescence, which led not only to increased GPC but also to increased grain yields; thus, this result further verified the improved NR from vegeta-

Very little research has been conducted on the accumulation of phenolic acids. Ma et al. [39] reported five key enzymes, namely phenylalanine ammonia lyase (PAL), coumaric acid

feic acid/5-hydroxyferulic acid O-methlytransferase (COMT), which are essential for the biosynthesis of phenolic acids. Ma et al. [39] also characterized gene expression patterns of nine candidate genes associated with phenolic acid biosynthesis during early and late grain filling stages, the most crucial growth stage in polyphenol accumulation [85, 86]. The study revealed that seven genes (*TaPAL1*, *TaPAL2, Ta4CL1, Ta4CL2, TaCOMT1, TaCOMT2* and *TaC3H2*) are highly expressed during the early stages of grain development among white, red and purple wheat. However, *TaC3H1* was the single gene that was expressed only during the later stage

H), 4-coumarate CoA ligase (4CL) and caf-

grain filling, acting as major regulators and executors in wheat and barley [81].

*3.3.1. Genes involved in micronutrient accumulation*

354 Wheat Improvement, Management and Utilization

tive organs to growing grain in transgenic lines [84].

H), cinnamic acid 4-hydrolase (C<sup>4</sup>

*3.3.2. Genes involved in phenolic acid accumulation*

3-hydrolase (C3
