The Synthesis and Regulation of Micronutrients in Rice Grains

Amino acids, vitamins and lipids are the important micronutrients in rice grains. Their synthesis and regulation have important effects on the normal growth and development in rice seeds. This review has mainly summarized the new advances in the synthesis and regulation of amino acids, vitamins and lipids in rice grains. Simultaneously, the challenges of the synthesis and accumulation of the micronutrients in rice grains were also discussed. This review provides important information for genetic improvement of grain quality in rice and, potentially, other staple cereals.


The Synthesis and Regulation of Amino Acids in Rice Grains
Amino acids play a very important role in plant growth and metabolism [20]. The contents of lysine and tryptophan in grains are low, and they are the first and second restricted essential amino acids in rice respectively. The content of lysine is also a relatively low essential amino acid in other main grain crops (such as corn, wheat, etc.), thus lysine is considered as the first limiting amino acid in cereal crops [21]; Ufaz and Galili [22]. In order to meet the needs of human nutrition balance, it is necessary to improve the protein content and the relative content of amino acids in rice grain. Through the aspartic acid metabolism pathway, not only lysine but also the other three essential amino acids, methionine, threonine and isoleucine could be synthesized in rice [23]. Aspartic kinase (AK), dihydropyridine dicarboxylic acid synthase (DHDPS) and lysine ketoglutarate reductase/yeast aminoate dehydrogenase (LKR/SDH) are the three key enzymes in aspartic acid metabolism pathway [24]. Among them, AK and DHDPS play a feedback inhibitory role in aspartic acid metabolism pathway. AK catalyzes the first step of lysine synthesis pathway, while DHDPS catalyzes the first step of dihydropyridine dicarboxylic acid branching. At the same time, lysine and threonine can feedback the inhibition of AK while lysine is the regulatory inhibitory factor of DHDPS Vidal, et al. [25]. When the content of lysine in grains is high, the activity of LKR/SDH will increase correspondingly, which will promote the degradation process of lysine in vivo and lead to the low content of lysine in grains [26]; [27]. A large number of studies based on aspartic acid metabolism pathway have indicated that if the expression of AK and DHDPS genes was promoted or the expression of LKR/SDH genes and 13-kDa prolamine genes was inhibited, the lysine content in rice plants was significantly increased [28]; [29]. If the lysine-rich exogenous protein was specifically expressed in rice grains or the RLRH1 and RLRH2 genes were overexpressed, the lysine content in rice grains could also be significantly increased [30]; [31]. Therefore, the specific quantitative expression of the key genes in the lysine metabolism pathway can greatly enhance the lysine content in rice grains.
Tryptophan and phenylalanine, are essential amino acids which can not be synthesized by the human and animal bodies.They play an important role in the growth, development and metabolism of both human and animal [32]. Tryptophan and phenylalanine belong to aromatic amino acids which are the precursors of various secondary metabolites in rice. They are closely related to the growth and development of rice and even the rice grain quality [33]. Therefore, increasing the content of tryptophan and phenylalanine in rice grain is of great significance to the improvement of nutritional quality of rice. Cuurently, there are many studies on increasing the content of lysine and methionine in rice grain while those aiming at trying to increase the content of tryptophan and phenylalanine in rice are relatively less [34].
Aromatic amino acids belong to the metabolic pathway of shikimic acid in plants, bacteria and fungi (Fig. 1), and they share a common precursor, branching acid. Various alpha-subunit-related genes of feedback-insensitive oaminobenzoic acid synthase (AS) have been used in the genetic improvement of tryptophan in crops [33]; [35]. In order to improve the content of tryptophan, a large number of corresponding mutants had been used, but it was difficult to find the mutants with significant changes in phenylalanine content [36]; [37]. The dehydratase ADT/PDT encoded by Mtr 1 could catalyze the last step of phenylalanine biosynthesis in the overexpressing of transgenic plants [38], and the contents of tryptophan and phenylalanine increased significantly, suggesting that oaminobenzoic acid synthetase and ADT/PDT dehydratase play a key role in regulating the metabolism of tryptophan and phenylalanine in rice grains. Cysteine and methionine are important amino acids that constitute proteins and the synthesis of the former (cysteine) can enhance the antioxidant stress ability in plants. Most other sulfur metabolites are directly or indirectly derived from cysteine, thus cysteine is at the center of sulfur metabolism in plants [39]; [40]. Serine acyltransferase and 3-phosphoglycerate dehydrogenase are two rate-limiting enzymes in the process of cysteine biosynthesis. Hydrogen sulfide and O-acetylserine (OAS) finally react with cysteine synthase (OAS-TL) to form cysteine [40]. Two rate-limiting enzymes (serine acyltransferase and glycerol 3-phosphate dehydrogenase) strictly regulate the catalytic reaction of cysteine synthesis resulting in the low content of cysteine. The synthesis of cysteine synthase complex (CSC), using cysteine synthase (OAS-TL) and serine acetyltransferase (SAT) will be more effective in regulating the process of cysteine biosynthesis [41]. Methionine is an essential amino acid that humans and animals can't synthesize themselves. Methionine as one of the essential protein source provides active methyl groups for the body, and it can also be converted into cysteine in vivo [42]; [43];. The lack of methionine can cause a variety of hazards to humans and livestock, and long-term consumption of foods with low methionine content may lead to many diseases. For example, methionine deficiency in livestock's diet will lead to the decrease of wool in sheep, milk production and meat quality in cows. The absence of methionine human diet will affect the absorption and utilization of other related amino acids by the body [44]. Therefore, increasing the content of methionine has been one of the important goals pursued by plant geneticists and breeders [33]. The expression of serine acetyltransferase gene was driven by a promoter with ubiquitin, which increased methionine and cysteine in rice by 1.4 times and 2.4 times respectively [45]. Simultaneously, the contents of leucine and valine also increased significantly, indicating that methionine could be transformed into isoleucine in rice. Therefore, the use of genetic engineering strategy can significantly improve the content of essential amino acids in rice grain, and then improve the nutritional quality of rice.

The Synthesis and Regulation of Lipid in Rice Grains
Lipid, including fat and phospholipids, is a very important nutrient in rice grains. It is mainly distributed in the aleurone layer outside the embryo and endosperm of rice seeds where a complex of lipids and amylose in rice grains form Xu, et al. [46]; Goufo, et al. [47]. At present, many QTLs have been located in rice genome and are closely related to lipids in their grains. However, the isolation and cloning of QTL related genes are rare Shao and Bao [48]. Fatty acid oxidase (LOX) is an important factor leading to the decline of rice nutritional quality, because it can catalyze the oxidation of lipids [23]. Both LOX-1, LOX-2, LOX-3 and r9-LOX-1 in rice genome encode fatty acid oxidase [49]; [50]. It was found that LOX-2, LOX-3 or r9-LOX-1 could inhibit the degradation of fatty acids. The degradation of β-carotene in golden rice could be effectively reduced by decreasing the expression of LOX-3 or r9-LOX-1.
Very long chain polyunsaturated fatty acid (VLCPUFAs) and long chain polyunsaturated fatty acid (LCPUFA) are essential regulatory substances for the synthesis and transport of cholesterol and eicosanoid [51]. They are the main components of nerve cells (such as the brain and retina) [52]; which in turn affect the development and health of the human body. Ultra long chain polyunsaturated fatty acids can be synthesized through different pathways (e.g. ω -6 metabolic pathway or ω-3 metabolic pathway) [53]. Therefore, proteins or enzymes encoded by many genes can improve the level of ultra long chain polyunsaturated fatty acids such as FAD3, D5 prolongase gene, ω-3 fatty acid desaturase gene, Δ 8-desaturase gene and Δ 5-desaturase gene [54]; [55]; [56]; [57]. FAD3 protein can catalyze the synthesis of α-linolenic acid in rice grains, and then it can be used to increase the content of α-linolenic acid in rice. α-linolenic acid is an important precursor of long-chain ω3-unsaturated fatty acids, and its content in rice grain is relatively low. If the FAD3 gene is over-expressed, the content of α-linolenic acid in rice grains can be greatly increased [58]. Although three FAD3 genes have been cloned in rice, it is unclear how these genes play a role in increasing the concentration of α-linolenic acid in rice grains.
Oil protein is abundant in the oil body of plant seeds and can be used to regulate the content of fat in seeds. The over-expression of soybean oil body protein gene was driven by rice endosperm-specific promoter, the fat content in transgenic rice grains increased by more than 36%, however, the total triglyceride fatty acid content did not change significantly [59]. Rice oil contains a large number of antioxidant substances such as oryzanol, lecithin, tocopherol and fertility trienol, which are beneficial to human health Choi, et al. [60]. The expression of GmFAD3-1 and OsFAD3 genes was driven by rice embryo-specific promoter REG, which resulted in a significant increase in αlinolenic acid content in rice embryos and aleurone layers. The increased α-linolenic acid is located in the sn-2 position of triglyceride and is easily digested and absorbed by human body Yin, et al. [61]. Previous studies have shown that OsLTP36 encoded a lipid transporter gene in rice, and if OsLTP36 gene was down regulated, it would seriously affect the development of rice seeds, and could significantly reduce the lipid content in rice grains [62]; [63]. The FAD3 gene of Brassica napus was specifically expressed in rice, which could significantly increase the content of C18: 3 fatty acids and improve the nutritional quality of rice [58]. At present, although important progress has been made in the study of lipid metabolism and some genes related to lipid metabolism in rice have also been isolated and cloned, how to regulate lipid metabolism pathway in rice grains remains to be further studied.

The Synthesis and Regulation of Vitamin in Rice Grains
The low levels of vitamin A in the endosperm of rice grains cause more than 250 million people in the world who rely on rice as their staple food to experience varying levels of vitamin A deficiency, which could lead to immune system-related diseases, or even permanent blindness [64]. Carotenoids, mainly beta-carotenoids are easily converted into the precursors to synthesize vitamin A in humans [65]. At present, genes related to the pathway of carotenoid biosynthesis have been successfully isolated and cloned, and these genes are widely found in bacteria, fungi and plants [66]. Although rice can synthesize carotenoids in leaves, some enzymes in the pathway of carotenoid synthesis are not expressed in the endosperm of rice. After genetic engineering modification, rice can produce a lot of β-carotenoids in endosperm and form yellow "Golden Rice". The first generation of Golden Rice contains 1.6 μg carotenoid per gram of dry weight of rice grain, which is equivalent to 100 μg of retinol in 300 g of rice per day, and it will greatly alleviate the symptoms of vitamin A deficiency in children [67]; [68]. By using maize octahydrolycopene synthase gene (psy) instead of narcissus octahydrolycopene synthase gene in the second generation of Golden Rice, the carotenoid content in rice grain could reach 37 μg.g -1 [69]. It is feasible to increase the β-carotene level in different indica and japonica rice varieties [66]; [70], but there is still a long way to go for the commercialization of golden rice.
The content of thiamine or vitamin B1 in rice grains is relatively low, with only about 18% of the recommended daily consumption content, thus vitamin B1 should be supplemented by other ways. Rice aleurone layer and embryo (containing more thiamine than endosperm) has been removed in the process of grain polishing, which leads to the further decrease of thiamine content in milled rice (mainly endosperm). Therefore, the lack of thiamine may cause beriberi in the population with milled rice as the only staple food [71]. In recent years, important progress has been made in the biosynthetic pathway of thiamine in plants [72]; [73], the synthesis of thiamine occurs in plastids. Under the action of thiamine phosphate (TMP) synthase, the two groups of pyrimidine and thiazole are condensed to form TMP, and hydrolysis of TMP into thiamine in cytoplasm and conversion to thiamine pyrophosphate (TPP). TPP produced by this pathway can bind to the precursor of thiamine biosynthetic gene and interfere with its gene expression, and regulating the ribosomal switch by RNA sequence. Interestingly, TPP ribosomal switch sequence located in the 3'-UTR region of THIC gene plays a negative regulatory role in thiamine biosynthesis [74]; [75]. The contents of TMP and TPP increased significantly in the leaves of plants with overexpression of THIC gene [76]. However, further research is needed to increase the content of thiamine in the endosperm of rice grains.
Folic acid, also known as vitamin B9 plays an important role in human growth and development. Lack of folic acid in diet can lead to many diseases [77]. Many genes have been found to be involved in the synthesis and metabolism of folic acid in plants such as GTP cyclase gene (GTPCHI), aminodeoxysynthase gene (ADC), hydroxymethyl dihydropurine gene (HMDHP), pyrophosphokinase gene (HPPK) and dihydrocharidase synthase gene (DHP) etc [78]; [79]. The over-expression of ADCS gene will increase the content of benzoic acid in plants, but the increase of benzoic acid will seriously inhibit the biosynthesis of folic acid, and ultimately lead to the decrease of folic acid content [80]. There was no significant change in folic acid content in transgenic plants with the overexpression of GTPCHI gene, but it should increase the folic acid content in transgenic plants to 50-100 times than the original level when GTPCHI gene and ADCS gene were overexpressed at the same time [81]. Using the promoter from maize driven barley gene HPPK/DHPS to express in rice, it was found that the content of folic acid in transgenic rice seeds reached about 1.5 times of the original level [77]. Therefore, through the strategy of metabolic engineering, using the genes related to folic acid biosynthesis pathway in close related species of rice, it can also increase the content of folic acid in rice grains.
Vitamin E is a compound that includes tocopherol and reproductive trienol family members in eight forms of fat-soluble antioxidants. As an important part of human defense, vitamin E provides anti-oxidative damage protection, thus reducing the occurrence of various diseases. The overexpression of hydroxyl pyruvate dioxygenase gene (HPPD) from Arabidopsis thaliana has no obvious effect on the content of tocopherol, but it can produce more reproductive tocotrienols. Finally, the activity of vitamin E in rice grains increased significantly [82]. In rice genome, at least 7 genes, such as MT1/2, OsHGGT, OsMPBQ, OsHPPD, OsTC, OsHPT and OsTMT, have been found to be involved in the synthesis of vitamin E in rice seeds [83]; [84]. The expression level of OsTMT gene and its alleles was closely related to the content of alpha-tocopherol in rice grains [65]. Overexpression of OsHPT gene can also increase the content of vitamin E components [85]. If GE gene mutation occurs, it can enhance the expression of genes related to vitamin E synthesis and metabolism, and then increase the content of vitamin E in rice grains [83]. Interestingly, over-expression of GmTMT2a resulted in a significant increase in the content of alphatocopherol [86], which provided a new idea for increasing the content of alpha-tocopherol in rice grains.
Vitamin C, also known as ascorbic acid, is an important water-soluble vitamin with antioxidant, antiatherosclerosis, improving immunity and anti-cancer properties. However, its content in rice grains is also very low [87]. Humans are unable to synthesize this important vitamin due to the lack of glycosolactone oxidase. Although the pathway of vitamin C biosynthesis and its associated genes are relatively clear in plants but there is little known about them in monocotyledonous plants especially in rice [33]. If the over-expression of the genes from Arabidopsis thaliana involved in vitamin C synthesis and metabolism (such as AtGGP, AtGDH, AtGME, AtGPP, AtGMP and AtGalLDH), the content of vitamin C in rice grains could be significantly increased [88]. Mutations in the genes related to vitamin C synthesis and metabolism in rice will not only reduce the content of vitamin C in the grains [89], but also have an important impact on the stress resistance and the development of the whole plant in rice. Therefore, in order to solve the problem of low vitamin content in rice grains, an increase in the vitamin content of rice seeds and improved nutritional quality, rice must make full use of its genes. To achieve this, we can also consider the introduction of exogenous genes, metabolic engineering, genetic engineering and other modern technical methods.

The Challenge and Prospect of Nutrient Improvement in Rice Grains
The nutrients of rice grains include starch, storage proteins, lipids, amino acids, vitamins and so on are improved by genetic engineering or metabolic engineering [90]. To produce the best functional proteins or enzymes driven by appropriate promoters of the target gene and promote the synthesis of macronutrients and micronutrients in rice grains, the growth and development of plants and other metabolic pathways need not to be affected [91]. At present, the biosynthetic pathway and regulation mechanism of some nutrients in rice are still unclear, this in turn limits the application of genetic engineering or metabolic engineering for genetic improvement in rice. Nutrients in rice grains have been researched extensively and important progress has been made by using the strategies of multi-omics (proteomics, metabolomics, transcriptome, etc.). For example, some undesirable allergic proteins improved by genetic engineering in rice can be screened by proteomics [92], and multiple metabolites related to abiotic stress resistance and nutritional starvation can be accurately quantified and identified in rice [93]; [94]. It is especially helpful to promote the accumulation of beneficial nutrients in seeds, which will be of great significance to improve human health.
At present, gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis mass spectrometry (CE-MS), X-ray fluorescence spectrometry (XRF), energy dispersive X-ray spectrometry (EDX), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as well as single cell imaging (SCIM) have developed rapidly in recent years [1]. All of these will provide new technical solutions for further research on the synthesis and accumulation of nutrients in rice grains. However, genetic improvement in rice grains is still challenging in view of the multiple steps in the process of simultaneous action of multiple genes on biosynthetic pathways or the multiple traits of multiple biosynthetic pathways in rice. Although many genes related to the synthesis of nutrients in rice grains have been revealed, functional genes are often directly or indirectly regulated by other genes [33], and there is a common phenomenon that the genes may have multiple effects. Therefore, functional genes that can be widely used ingrain nutrient genetics improvement and breeding are relatively rare in rice. In order to promote the synthesis and accumulation of nutrients in rice grains, these functional genes often need to develop specific, tissue-specific or inducible promoters to drive their expression.
In recent decades, genome editing technology based on sequence-specific nucleases (SSNs) has developed rapidly, and has become one of the most effective new tools for rice genetic improvement [95]; [96]. Particular attention should be given to CRISPR/Cas9 technology, which has many advantages: (1) Editing target genes accurately; (2) There is no need for hybridization and backcross, and it is convenient and fast; (3) No need for large capital investment; (4) Individuals without selection markers can be obtained [96]; [97]. Therefore, CRISPR/Cas9 technology has been widely used in rice nutrition biosynthesis and metabolism researches [98]; [99]. Therefore, genome editing technology, represented by CRISPR/Cas9 technology will play a more and more important role in the process of grain nutrition genetic improvement and breeding of new varieties in rice. This will eventually greatly accelerate the genetic improvement of rice grain quality.