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The Genetic Basis and Nutritional Benefits of Pigmented Rice Grain

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The Genetic Basis and Nutritional Benefits of Pigmented Rice Grain

Abstract and Figures

Improving the nutritional quality of rice grains through modulation of bioactive compounds and micronutrients represents an efficient means of addressing nutritional security in societies which depend heavily on rice as a staple food. White rice makes a major contribution to the calorific intake of Asian and African populations, but its nutritional quality is poor compared to that of pigmented (black, purple, red orange, or brown) variants. The compounds responsible for these color variations are the flavonoids anthocyanin and proanthocyanidin, which are known to have nutritional value. The rapid progress made in the technologies underlying genome sequencing, the analysis of gene expression and the acquisition of global ‘omics data, genetics of grain pigmentation has created novel opportunities for applying molecular breeding to improve the nutritional value and productivity of pigmented rice. This review provides an update on the nutritional value and health benefits of pigmented rice grain, taking advantage of both indigenous and modern knowledge, while also describing the current approaches taken to deciphering the genetic basis of pigmentation.
| Secondary metabolism in rice. (A) A schematic representation of the shikimic acid pathway. DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DAHPS, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; DHQ/SDH, 3-dehydroquinate dehydratase/shikimate 5 dehydrogenase; DHQS, 3-dehydroquinate synthetase; DHS, 3-dehydroshikimic acid; SDH, shikimate dehydrogenase; SK, shikimate kinase; S3P, shikimic acid 3-phosphate; EPSPS, 5-enolpyruvylshikimate 3-phosphate synthase; EPSP, 5-enolpyruvylshikimate 3-phosphate; CS, chorismate synthase; CM, chorismate mutase; PAT, prephenate aminotransferase; ADT, arogenate dehydratase; PDT, prephenate dehydratase; PPY-AT, phenylpyruvate aminotransferase (Tzin and Galili, 2010; Widhalm and Dudareva, 2015; Santos-Sánchez et al., 2019). (B) Possible routes to the production of benzoic acid, benzoic acid-derived compounds and lignin. CNL, cinnamate-CoA ligase; CHD, cinnamoyl-CoA-dehydrogenase/hydratase; KAT1, 3-ketoacyl-CoA thiolase; TE, CoA thioesterase; BA2H, benzoic acid 2-hydroxylase; BALDH, benzaldehyde dehydrogenase; AO, aldehyde oxidase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; ICS, isochorismate synthase; CCR, cinnamoyl-CoA reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; F5H, ferulate 5-hydroxylase; CSE, caffeoyl shikimate esterase; COMT, caffeic acid O-methyltransferase; CAD, cinnamyl alcohol dehydrogenase; LAC, laccase; POD, peroxidase; p-HBD, p-hydroxybenzaldehyde; HBDS, 4-hydroxybenzaldehyde synthase; HCHL, 4-hydroxycinnamoyl-CoA hydratase/lyase; HBD, 4-hydroxybenzaldehyde dehydrogenase (Qualley et al., 2012; Gallage and Møller, 2015; Widhalm and Dudareva, 2015; Liu et al., 2018). (C) Flavonoid metabolism. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthetase; CHI, chalcone isomerase; F3 H, flavone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanin synthase; ANR, anthocyanin reductase; GT, glucosyltransferase; LAR, leucoanthocyanidin reductase; MT, O-methyltransferase; F2H, flavanone 2-hydroxylase (Chen et al., 2013; Galland et al., 2014). The square dot arrows indicates steps which have not yet been fully elucidated, while the black arrows indicate steps supported by genetic evidence.
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REVIEW
published: 13 March 2020
doi: 10.3389/fgene.2020.00229
Edited by:
Manish Kumar Pandey,
International Crops Research Institute
for the Semi-Arid Tropics (ICRISAT),
India
Reviewed by:
Larry Parnell,
Jean Mayer USDA Human Nutrition
Research Center on Aging at Tufts
University, United States
Yue-ie Caroline Hsing,
Academia Sinica, Taiwan
*Correspondence:
Nese Sreenivasulu
n.sreenivasulu@irri.org
Specialty section:
This article was submitted to
Nutrigenomics,
a section of the journal
Frontiers in Genetics
Received: 20 May 2019
Accepted: 26 February 2020
Published: 13 March 2020
Citation:
Mbanjo EGN, Kretzschmar T,
Jones H, Ereful N, Blanchard C,
Boyd LA and Sreenivasulu N (2020)
The Genetic Basis and Nutritional
Benefits of Pigmented Rice Grain.
Front. Genet. 11:229.
doi: 10.3389/fgene.2020.00229
The Genetic Basis and Nutritional
Benefits of Pigmented Rice Grain
Edwige Gaby Nkouaya Mbanjo1,2 , Tobias Kretzschmar3, Huw Jones4, Nelzo Ereful4,
Christopher Blanchard5, Lesley Ann Boyd4and Nese Sreenivasulu1*
1International Rice Research Institute, Los Baños, Philippines, 2International Institute for Tropical Agriculture, Ibadan, Oyo,
Nigeria, 3Southern Cross Plant Science, Southern Cross University, Lismore, NSW, Australia, 4National Institute
of Agricultural Botany, Cambridge, United Kingdom, 5School of Biomedical Sciences, Charles Sturt University, Wagga
Wagga, NSW, Australia
Improving the nutritional quality of rice grains through modulation of bioactive
compounds and micronutrients represents an efficient means of addressing nutritional
security in societies which depend heavily on rice as a staple food. White rice makes
a major contribution to the calorific intake of Asian and African populations, but its
nutritional quality is poor compared to that of pigmented (black, purple, red orange, or
brown) variants. The compounds responsible for these color variations are the flavonoids
anthocyanin and proanthocyanidin, which are known to have nutritional value. The rapid
progress made in the technologies underlying genome sequencing, the analysis of gene
expression and the acquisition of global ‘omics data, genetics of grain pigmentation has
created novel opportunities for applying molecular breeding to improve the nutritional
value and productivity of pigmented rice. This review provides an update on the
nutritional value and health benefits of pigmented rice grain, taking advantage of both
indigenous and modern knowledge, while also describing the current approaches taken
to deciphering the genetic basis of pigmentation.
Keywords: pigmented rice grain, nutrition, flavonoids, metabolites, genetics
INTRODUCTION
Rice is a staple food for over half of the world’s population (World Rice Production, 2019). Meeting
the demand of future rice supply for the growing population, which has been predicted to reach
9.7 billion by 20501, is central for ensuring food and nutritional security. In addition to its critical
importance to Asian populations as a source of food, rice also features in a range of social, cultural,
economic, and religious activities (Ahuja et al., 2007;Hedge et al., 2013;Sathya, 2013). In sub-
Saharan Africa the consumption of rice is projected to grow from its current level of 27–28 Mt
per year to around 36 Mt by the end of 2026 (Terungwa and Yuguda, 2014;Nigatu et al., 2017),
replacing some of the current demand for cassava, yam, maize, millet, and sorghum.
Most of the nutrients found in rice grain accumulate in the outer aleurone layer and embryo,
the endosperm being composed primarily of starch. The process of dehulling and milling discards
most micronutrients, fatty acids, anti-oxidants, and fiber. As a result, diets over-reliant on white rice
risk deficiencies for several nutritional factors (Verma and Shukla, 2011;Sharma et al., 2013;Saneei
et al., 2016;Sarma et al., 2018). The focus of rice breeding has long been concentrated on improving
the crop’s productivity, although some emphasis has been given to improving the size, shape, and
amylose content of the grain (Breseghello, 2013;Rao et al., 2014). The nutritional quality of the
1https://www.un.org/development/desa/en/news/population/world-population- prospects-2019.html
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grain produced by certain traditional landraces has been shown
to be higher than that of the grain produced by conventional,
modern rice varieties, largely due to their more effective
accumulation of bioactive compounds (Bhat and Riar, 2015;
Berni et al., 2018). A growing consumer interest in health-
promoting food products is generating a substantial market
for more nutritionally valuable rice, creating health benefits
for the large number of people for whom rice is a staple,
while simultaneously generating economic benefits for the
producers (Terungwa and Yuguda, 2014). As a result, the focus
of a number of major rice research programs is turning to
the issue of nutritional quality, encompassing an improved
micronutrient and anti-oxidant content, along with a reduction
in the grains’ glycemic index.
This review explores the nutritional and health attributes of
pigmented rice grain, based on both indigenous knowledge and
current research, and discusses the potential of pigmented rice
grain to address nutritional food security. In addition, it explores
the genetic basis of grain pigmentation, and suggests the potential
contribution which ‘omics technologies can make to address the
challenge of the double burden of malnutrition.
INDIGENOUS KNOWLEDGE,
COMPLEMENTED WITH
CORROBORATED SCIENTIFIC
EVIDENCE, INFORMS ON THE
POTENTIAL OF PIGMENTED RICE
GRAIN TO IMPROVE NUTRITION AND
HEALTH
Indigenous diets have developed to meet the needs of local
communities over a long period of time, and the knowledge
associated with these should be viewed as a resource to inform the
discussion concerning the place of rice in the modern diet (Berni
et al., 2018;Khatri, 2018). The value of such landraces in the
context of both human nutrition and health (Rahman et al., 2006;
Chunta et al., 2014) can be exemplified by the proven advantages
of consuming pigmented grain (Figure 1;Rahman et al., 2006;
Umadevi et al., 2012;Sathya, 2013). In particular, pigmented rice
has been associated with anti-inflammatory and diuretic activity
(Umadevi et al., 2012). Based on native indigenous knowledge,
it has also been recommended for the treatment of diarrhea,
vomiting, fever, hemorrhaging, chest pain, wounds, burns, and
gastrointestinal problems, as well as addressing various liver
and kidney disorders (Hedge et al., 2013;Sathya, 2013). Certain
pigmented rice varieties are still used to treat skin diseases, blood
pressure, fever, paralysis, rheumatism, and leucorrhea, and even
as the basis of a general health tonic (Ahuja et al., 2007). In
the Philippines, “tiki tiki,” derived from rice bran, has been used
to cure thiamine deficiency (Umadevi et al., 2012). In India,
the grain of pigmented rice landraces is offered to lactating
mothers, and is used to both treat jaundice and cure paralysis.
The rice variety “Laicha” was given its name because of its
ability to prevent an eponymous skin disease (Das and Oudhia,
2000). For more than 2,000 years, grain of the South Indian
FIGURE 1 | Genetic variation for grain pigmentation in rice. Grains featuring
(a,b) white, (c,d) brown, (e–h) purple, (i,j) dark purple or black, (k–n) red, and
(o,p) mixed colored pericarp. The application of various genomic approaches
to understand the genetic pathway of grain pigmentation is outlined.
landrace “Kavuni” has been reported to exhibit anti-oxidant, anti-
arthritic, and anti-diabetic properties, and has been used to cure
gastritis and peptic ulcers, as well as to enhance blood circulation
(Valarmathi et al., 2014;Hemamalini et al., 2018).
A number of scientifically based studies have provided
evidence to support the hypothesis that pigmented rice grain
possesses anti-oxidant, anti-diabetic, anti-hyperlipidemic, and
anti-cancer activity (Baek et al., 2015;Boue et al., 2016), which
is reviewed below.
Anti-oxidant Activity
Dietary anti-oxidants represent an effective means of combating
the accumulation of harmful reactive oxygen species and of
balancing the redox status of the body (Krishnanunni et al., 2014).
Analysis of extracts made from pigmented rice grain has shown
that the phenolic compounds tocopherol and anthocyanin are
efficient neutralizers of reactive oxygen species (Zhang et al.,
2015;Ghasemzadeh et al., 2018), while animal tests have proven
that these compounds are bioavailable (Tantipaiboonwong et al.,
2017). Several studies have shown that the elevated anti-oxidation
activity exhibited by pigmented rice grains (most markedly by
black rice) can be used to mitigate the inflammatory response
(Chakuton et al., 2012;Petroni et al., 2017).
Anti-diabetic Activity
The grain of some traditional pigmented rice varieties have
proven to be effective in supporting glucose homeostasis, and are
thus useful for the management of diabetes mellitus (Hemamalini
et al., 2018). Unlike white rice grain consumption, which
raises blood glucose levels, consuming pigmented grain can
reduce blood glucose levels. Extracts of pigmented rice grain
and bran have been shown to effectively inhibit the activity
of endogenous α-amylase and α-glucosidase, thereby inhibiting
the conversion of starch to glucose in the small intestine,
which acts as a source of resistant starch to be utilized by
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gut microbiota in the colon (Boue et al., 2016;Chiou et al.,
2018). While extracts made from both red and purple grain have
been reported to inhibit α-glucosidase activity, only the former
was effective in also inhibiting α-amylase activity (Valarmathi
et al., 2014;Boue et al., 2016). The anthocyanins found in
the whole grain of black rice acted as a potent inhibitor of
β-glucosidase, thus delaying the absorption of carbohydrates
(Chandramouli et al., 2018). Extracts of black rice bran have
also been shown to induce the repair and regeneration of
pancreatic beta cells (Wahyuni et al., 2016). Overall, the anti-
diabetic effects of pigmented rice seem to arise from a synergistic
effect of anthocyanin, proanthocyanidin, vitamin E, γ-oryzanol,
and various flavonoids (Tantipaiboonwong et al., 2017). Black
rice extracts reduced blood glucose levels more quickly than
did extracts from red rice, a difference which was attributed
to the presence of cyanidin 3-glucoside, a compound which
activates insulin sensitivity, glucose uptake, and adiponectin
secretion (Matsukawa et al., 2016;Tantipaiboonwong et al.,
2017). However, many of the black rice are low in its amylose
content and upon milling most of the anthocyanins accumulated
in aleurone will be lost, thus not necessarily would possess
low GI property when consumed in the form of milled rice
(Kang et al., 2011).
Anti-cancer Activity
A considerable body of evidence suggests that consumption of
pigmented rice has a protective effect against certain cancers.
Ghasemzadeh et al. (2018) demonstrated that extracts of both
black and red rice inhibit the proliferation of breast cancer cells.
The phenolic acids, flavonoids, anthocyanins, and phytic acid
present in extracts of purple rice bran have been shown to act
as anti-mutagens and potential suppressors of cancer. It has
been proposed that these phytochemicals act by either blocking
the carcinogenetic cytochromes P450 CYP1A1 and CYP1B1
and/or by effectively scavenging free radicals (Insuan et al.,
2017). Bioactive compounds of pigmented grains can reduce the
viability of cancer cells and even induce their apoptosis. The
mechanistic basis of this effect has been found to be variety-
dependent, reflecting differences in the spectrum of bioactive
compounds present in each rice variety (Baek et al., 2015). The
high anthocyanin content of purple rice has been associated
with an inhibitory effect on the growth of human hepatocellular
carcinoma cells (Banjerdpongchai et al., 2013), while extracts of
purple rice bran were able to block the first stage of aflatoxin
B1-initiated hepatocarcinogenesis by inhibiting key metabolic
activating enzymes (Suwannakul et al., 2015). Extracts of red rice
have been shown to limit the invasiveness of cancer cells in a
dose-dependent manner (Pintha et al., 2014). The phytosterols
24-methylenecycloartanol, β-sitosterol, gramisterol, campesterol,
stigmasterol, cycloeucalenol, 24-methylene-ergosta-5-en-3β-ol,
and 24-methylene-ergosta-7-en-3β-ol, all of which are present
in extracts of black rice bran, have also been reported to be
effective as agents restricting the proliferation of murine leukemic
cells (Suttiarporn et al., 2015). Consequently, one of the long-
term strategies proposed by Luo et al. (2014) to prevent breast
cancer metastasis relies on the inclusion of pigmented rice
in the human diet.
THE BIOCHEMICAL PROPERTIES OF
PIGMENTED RICE GRAIN
Phytosterols, Carotenoids, Vitamins, and
Micronutrients in Pigmented Rice Grain
Phytosterols
Rice grains contain a wide range of secondary metabolites
(Table 1). Pigmented grain appears to accumulate a higher level
of γ-oryzanol than does non-pigmented grain (Chakuton et al.,
2012). The grain accumulates the active anti-oxidant γ-oryzanol,
which comprises a mixture of several phytosteryl ferulates
(Chakuton et al., 2012), in particular 24-methylenecycloartanyl
ferulate, cycloartenyl ferulate, campesteryl ferulate, and
β-sitosteryl ferulate (Zubair et al., 2012;Pereira-Caro et al.,
2013). The most important nutritional benefit of the phytosterols
is their ability to both inhibit the absorption of cholesterol
and to control the blood’s content of undesirable lipoproteins
(Jesch and Carr, 2017). The predominant phystosterols detected
in commercial rice varieties are β-sitosterol, followed by
campesterol, 15-avenasterol, and stigmasterol (Zubair et al.,
2012). The bran of the black rice variety “Riceberry” also
harbors three additional sterols, namely 24-methylene-ergosta-
5-en-3β-ol, 24-methylene-ergosta-7-en-3β-ol, and fucosterol
(Suttiarporn et al., 2015).
Carotenoids
Carotenoids represent another class of nutritionally beneficial
compounds (Roberts and Dennison, 2015). Lutein and
zeaxanthin represent together >90% of the carotenoids
produced by rice, with carotenes, lycopenes, and β-carotene
present in trace amounts (Pereira-Caro et al., 2013;Melini and
Acquistucci, 2017). Most of this class of compound is present in
the bran, with little or no carotenoids being found in milled rice
(Petroni et al., 2017). Grain carotenoid content is a genetically
variable trait, and is strongly correlated with grain pigmentation
(Ashraf et al., 2017). Red and black rice accumulate a particularly
high carotenoid content, while white rice accumulates very little
(Ashraf et al., 2017;Petroni et al., 2017).
Vitamins
Rice grain represents a good source of vitamin E, including both
the tocopherols and the tocotrienols (Zubair et al., 2012). The β-
and γ-tocotrienols are the most abundant forms present in rice
(Irakli et al., 2016). According to Gunaratne et al. (2013), red rice
grains harbor higher levels of total tocopherol and tocotrienol
than do the grains of modern white rice varieties. Note, however,
that dehulling and milling strongly reduce the tocopherol content
of the grain (Zubair et al., 2012).
Micronutrients
Rice grain contains traces of a number of essential
micronutrients, namely zinc, magnesium, iron, copper,
potassium, manganese, and calcium (Table 1;Raghuvanshi
et al., 2017;Shozib et al., 2017;Shao et al., 2018). Some genetic
variation in mineral content has been reported; but in general,
pigmented rice grain accumulates higher amounts than does
white grain rice (Shozib et al., 2017). Other studies have
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TABLE 1 | Bioactive and nutritional compounds identified in pigmented rice.
Compound PubChem CID Compound class Figure 2aReferences
Cyanine 3-glucoside 197081 Anthocyanin XPereira-Caro et al., 2013;
Peonidin-3-glucoside 443654 Anthocyanin XZhang et al., 2015;
Cyanidin 128861 Anthocyanin Tantipaiboonwong et al., 2017;
Cyanidin-3,5-diglucoside 44256718 Anthocyanin XShao et al., 2018
Cyanidin-3-O-(6"-O-p-coumaroyl)glucoside Anthocyanin X
Pelargonidin-3-O-glucoside 443648 Anthocyanin
Peonidin-3-O-(6"-O-p-coumaroyl)glucoside Anthocyanin X
Cyanidin-3-O-arabidoside Anthocyanin X
Flavone 10680 Flavone
Luteolin-6/8-C-pentoside-6/8-C-hexoside (2 isomers) Flavone Pereira-Caro et al., 2013;
Apigenin-6/8-C-pentoside-8/6-C-hexoside (three isomers) Flavone glycoside Kim et al., 2014;
Apigenin-6-C-glucosyl-8-C-arabinoside Flavone Ghasemzadeh et al., 2018;
Tricin-O-rhamnoside-O-hexoside Flavone Poulev et al., 2019
Tricin 5281702 Flavone X
Chrysoeriol 5280666 Flavone X
Luteolin 5280445 Flavone X
Apigenin 5280443 Flavone X
Caffeic acid 689043 Hydrocinnamic acid XGunaratne et al., 2013;
p-Coumaric acid 637542 Hydrocinnamic acid XZhang et al., 2015;
Ferulic acid 445858 Hydrocinnamic acid XIrakli et al., 2016;
Sinapic acid 637775 Hydrocinnamic acid XTantipaiboonwong et al., 2017;
Isoferulic acid 736186 Hydrocinnamic acid Chiou et al., 2018;
Chlorogenic acid 1794427 Hydrocinnamic acid Ghasemzadeh et al., 2018;
Shao et al., 2018
2,5-Dihydroxybenzoic acid 3469 Hydroxybenzoic acid XKim et al., 2014;
p-Hydroxybenzoic acid 135 Hydroxybenzoic acid XValarmathi et al., 2014;
Gallic acid 370 Hydroxybenzoic acid XSuwannakul et al., 2015;
Vanillic acid 8468 Hydroxybenzoic acid XHuang and Lai, 2016;
Syringic acid 10742 Hydroxybenzoic acid XIrakli et al., 2016;
Protocatechuic acid 72 Hydroxybenzoic acid XTantipaiboonwong et al., 2017;
Salicylic acid 338 Hydroxybenzoic acid XGhasemzadeh et al., 2018;
β-Resorcylic acid 1491 Hydroxybenzoic acid Shao et al., 2018
Protocatechualdehyde 8768 Phenolic aldehyde Huang and Lai, 2016
8-50-Coupled diferulic acid Phenolic dehydrodimer Zhang et al., 2015
5-50-Coupled diferulic acid Phenolic dehydrodimer
8-80-Coupled diferulic acid benzofuran form Phenolic dehydrodimer
Proanthocyanidin dimer Proanthocyanin XGunaratne et al., 2013
Proanthocyanidin trimer Proanthocyanin X
Catechin 73160 Flavanonol XTantipaiboonwong et al., 2017;
Epicatechin 72276 Flavanonol XGhasemzadeh et al., 2018
Quercetin 5280343 Flavonol Pereira-Caro et al., 2013;
Quercetin-3-O-glucoside Flavonol Valarmathi et al., 2014;
Quercetin-3-O-rutinoside Flavonol Chiou et al., 2018;
Isorhamnetin-3-O-glucoside 5318645 Flavonol Ghasemzadeh et al., 2018;
Myricetin 5281672 Flavonol Poulev et al., 2019
Rutin 5280805 Flavonol
Kaempferol 5280863 Flavonol
Kaempferide 5281666 Flavonol
Naringenin 932 Flavanone XChiou et al., 2018
(Continued)
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TABLE 1 | Continued
Compound PubChem CID Compound class Figure 2aReferences
Cycloartenol ferulate 134695320 γ-Oryzanol Chakuton et al., 2012;
24-Methylenecycloartenol ferulate 9920169 γ-Oryzanol Gunaratne et al., 2013;
Campesteryl ferulate 15056832 γ-Oryzanol Pereira-Caro et al., 2013
β-Sitosteryl ferulate 9938436 γ-Oryzanol
17-Campesteryl ferulate γ-Oryzanol
Campestanyl ferulate 13786591 γ-Oryzanol
Sitostanyl ferulate 11227138 γ-Oryzanol
Phytic acid 890 Phytic acid Chakuton et al., 2012;Insuan
et al., 2017
Tocotrienols (α-, β-, γ-, δ-forms) 9929901 Vitamin E Zubair et al., 2012;
Tocopherols (α-, β-, γ-, δ-forms) 14986 Vitamin E Gunaratne et al., 2013;Irakli
et al., 2016
Riboflavin 493570 Vitamin B2 Valarmathi et al., 2014
Nicotinic acid 938 Vitamin B3 Kim et al., 2014
Lutein 5281243 Carotenoid Pereira-Caro et al., 2013;
Zeaxanthin 5280899 Carotenoid Valarmathi et al., 2014;
β-Carotene 5280489 Carotenoid Irakli et al., 2016;
Lycopene 446925 Carotenoid Melini and Acquistucci, 2017
β-Carotene-4,40-dione Carotenoid
all-trans-3,3’,4,4’-Tetrahydrospirilloxanthin 5366411 Carotenoid
100-Apo-β-carotenoic acid Carotenoid
24-Methylene-ergosta-5-en-3β-ol Phytosterol Suttiarporn et al., 2015
24-Methylene-ergosta-7-en-3β-ol Phytosterol
Fucosterol 5281326 Phytosterol
Gramisterol 5283640 Phytosterol
Campesterol 173183 Phytosterol
Stigmasterol 5280794 Phytosterol
β-Sitosterol 222284 Phytosterol
Cycloeucalenol 101690 Triterpenoid Suttiarporn et al., 2015
Lupenone 92158 Triterpenoid
Lupeol 259846 Triterpenoid
24-Methylenecycloartanol 94204 Triterpenoid
LysoPC 14:0 460604 Phospholipid Kim et al., 2014
LysoPC 18:2 11005824 Phospholipid
LysoPC 16:0 460602 Phospholipid
LysoPC 18:1 53480465 Phospholipid
Histidine 6274 Essential amino acid Kim et al., 2014;
Threonine 6288 Essential amino acid Thomas et al., 2015
Valine 6287 Essential amino acid
Methionine 6137 Essential amino acid
Lysine 5962 Essential amino acid
Isoleucine 6306 Essential amino acid
Leucine 6106 Essential amino acid
Phenylalanine 6140 Essential amino acid X
L-Aspartate 5460294 Non-essential amino acid Kim et al., 2014;
Serine 5951 Non-essential amino acid Valarmathi et al., 2014;
Glutamine 5961 Non-essential amino acid Thomas et al., 2015
(Continued)
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TABLE 1 | Continued
Compound PubChem CID Compound class Figure 2aReferences
Glycine 750 Non-essential amino acid
Arginine 6322 Non-essential amino acid
Alanine 602 Non-essential amino acid
Proline 614 Non-essential amino acid
Tyrosine 6057 Non-essential amino acid
α-Aminobutyric acid (AABA) 6657 Non-essential amino acid
Potassium (K) 5462222 Mineral Valarmathi et al., 2014;
Calcium (Ca) 5460341 Mineral Thomas et al., 2015;
Magnesium (Mg) 5462224 Mineral Raghuvanshi et al., 2017;
Sodium (Na) 5360545 Mineral Shozib et al., 2017;
Chromium (Cr) 23976 Mineral Hurtada et al., 2018
Iron (Fe) 23925 Mineral
Manganese (Mn) 23930 Mineral
Zinc (Zn) 23994 Mineral
Copper (Cu) 23978 Mineral
Phosphorus (P) 5462309 Mineral
Caproic acid 8892 Fatty acid Valarmathi et al., 2014;
Caprylic acid 379 Fatty acid Thomas et al., 2015
Capric acid 2969 Fatty acid
Lauric acid 3893 Fatty acid
Tridecanoic acid 12530 Fatty acid
Myristic acid 11005 Fatty acid
Pentadecanoic acid 13849 Fatty acid
Palmitic acid 985 Fatty acid
Stearic acid 5281 Fatty acid
Arachidic acid 10467 Fatty acid
9-Octadecanoic acid 965 Fatty acid
Undecanoic acid 8180 Fatty acid
Oleanolic acid 10494
Myristoleic acid 5281119 Mono-unsaturated fatty acid Valarmathi et al., 2014;
cis-10-Pentadecenoic acid 5312411 Mono-unsaturated fatty acid Thomas et al., 2015
Oleic acid 445639 Mono-unsaturated fatty acid
cis-Vaccenic acid 5282761 Mono-unsaturated fatty acid
Erucic acid 5281116 Mono-unsaturated fatty acid
Hexadecadienoic acid Polyunsaturated fatty acid Valarmathi et al., 2014;
Hexadecatrienoic acid 6506600 Polyunsaturated fatty acid Thomas et al., 2015
Linoleic acid 5280450 Polyunsaturated fatty acid
Octadecatetraenoic acid 11778225 Polyunsaturated fatty acid
cis-11,14,17-Eicosatrienoic acid 5312529 Polyunsaturated fatty acid
cis-5,8,11,14-Eicosatetraenoate acid Polyunsaturated fatty acid
Eicosatetraenoic acid 21863049 Polyunsaturated fatty acid
Pinellic acid 9858729 Oxylipin Kim et al., 2014
Succinic acid 1110 Carboxylic acid
Maleic acid 444266 Carboxylic acid
Malonic acid 867 Carboxylic acid
Citric acid 311 Carboxylic acid
Cinnamic acid 444539 Carboxylic acid X
D-Xylose 135191 Sugar Kim et al., 2014;
D-Fructose 2723872 Sugar Valarmathi et al., 2014
D-Glucose 5793 Sugar
Maltose 439341 Sugar
myo-Inositol 892 Sugar
aCompounds present in Figure 2.
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suggested that pigmented rice contains higher levels of zinc,
iron, and manganese than does white grain, but a lower level
of phosphorus (Raghuvanshi et al., 2017;Hurtada et al., 2018;
Shao et al., 2018). Brown rice can provide as much as 75% of the
recommended daily intake of zinc, copper, and iron, but this falls
to just 37% for white rice (Hashmi and Tianlin, 2016).
Flavonoid Metabolism in Pigmented
Rice Grain
The major flavonoids present in pigmented rice grain are
proanthocyanidins and anthocyanins (Table 1). The synthesis of
the flavonoids is initiated by the deamination of phenylalanine
to form cinnamic acid, a reaction catalyzed by phenylalanine
ammonia lyase. Cinnamate 4-hydroxylase catalyses the oxidation
of cinnamic acid to 4-coumaric acid, which is in turn converted
to 4-coumaroyl-CoA through the action of 4-coumaroyl-CoA
ligase (Cheng et al., 2014). The rate limiting step is the
conversion of cinnamic acid to p-coumaroyl-CoA, which affects
the synthesis of phenolic acids, flavanones, proanthocyanidins,
and anthocyanidins (Figure 2).
Phenolic Acids
Compared to white grain, pigmented grain contains a higher level
of phenolic acids (Gunaratne et al., 2013;Zhang et al., 2015).
Cinnamic acid serves as a precursor for the synthesis of various
phenolic acids, including p-coumaric acid, ferulic acid, sinapic
acid, isoferulic acid, and 2,5-dihydroxybenzoic acid (Zhang et al.,
2015;Shao et al., 2018). The predominant phenolic acids present
in white rice are p-coumaric acid and ferulic acid; these are largely
utilized as building blocks for lignin synthesis (Figure 2). In an
alternative pathway, particularly active in black rice, cinnamic
acid is converted to vanillic acid and protocatechuic acid (Zhang
et al., 2015;Shao et al., 2018). In red rice, caffeic acid has
been identified as a minor phenolic acid, while this compound
is not detectable in brown rice (Gunaratne et al., 2013;Zhang
et al., 2015;Irakli et al., 2016;Shao et al., 2018). Additional
phenolic acids identified include syringic acid in the extract of
brown, red, and black rice (Ghasemzadeh et al., 2018;Shao et al.,
2018), pinellic acids in red and white rice (Kim et al., 2014),
hydroxybenzoic acid in black rice extracts (Tantipaiboonwong
et al., 2017), and gallic acid in the extracts of the red rice mutant
AM-425 (Chiou et al., 2018). Four diferulic acids (phenolic acid
dehydrodimers) are present in the insoluble-bound (Table 1;
Zhang et al., 2015).
Flavanones
The condensation and subsequent intramolecular cyclization of
three molecules of malonyl CoA and one of 4-coumaroyl-CoA
is then catalyzed by chalcone synthetase to produce naringenin
chalcone. Naringenin chalcone is isomerized into naringenin by
the action of chalcone isomerase to form the flavones (Figure 2).
Small quantities of flavones and flavanol glycosides have been
detected in the grain, notably luteolin-6/8-C-pentoside-6/8-C-
hexoside and certain derivatives of apigenin (Table 1). In the
tricin pathway, a flavone synthase II enzyme converts naringenin
to apigenin, which is then converted first to luteonin by flavonoid
30-hydroxylase, and then to tricin by O-methyltransferase and
chrysoeriol 50-hydroxylase (Park et al., 2016;Figure 2). Apigenin,
luteolin, tricetin, tricin, quercetin, and myricetin have all been
detected in extracts of red and brown rice bran (Table 1;
Galland et al., 2014;Ghasemzadeh et al., 2018). The synthesis
of C-glycosylated flavanones begins with the conversion of
naringenin to 2-hydroxyflavanone by flavanone 2-hydroxylase,
which is then C-glycosylated by C-glucosyltransferase and
finally is dehydrated by an as yet unknown enzyme (Figure 2;
Du et al., 2010;Galland et al., 2014;Sasaki et al., 2014;
Park et al., 2016;Poulev et al., 2019). Other flavonoid-like
compounds identified in rice include quercetin-3-O-glucoside,
quercetin-3-O-rutinoside, methoxy-flavanol-3-O-glucoside, and
isorhamnetin-3-O-glucoside (Pereira-Caro et al., 2013;Kim
et al., 2014); tricin-O-rhamnoside-O-hexoside and apigenin-6-C-
glucosyl-8-C-arabinoside are particularly predominant in white
rice grains (Kim et al., 2014).
Proanthocyanidins
Proanthocyanidins are oligomers and polymers of flavan-
3-ols (Gunaratne et al., 2013). Naringenin, the universal
substrate for their synthesis, is 30-hydroxylated by flavonoid
30-hydroxylase, producing eriodictyol, which is then converted
to dihydroquercetin by the action of flavone 3-hydroxylase
(Figure 2). Dihydroflavonol 4-reductase catalyses the conversion
of dihydroquercetin into leucoanthocyanidins. Leucocyanidin is
converted into the flavan-3-ol catechin by leucoanthocyanidin
reductase, while catechin monomers are polymerized by a
yet unknown pathway to form proanthocyanidin (Figure 2;
Zhao et al., 2010;Galland et al., 2014). Proanthocyanidins
and catechins make up the bulk of the phenolic compounds
found in red rice, being responsible for the red pigmentation
of the pericarp (Pereira-Caro et al., 2013;Kim et al., 2014). No
proanthocyanidins have been detected in white rice accessions
(Gunaratne et al., 2013), while some black rice varieties have been
reported to contain them (Vichit and Saewan, 2015).
Anthocyanidins
The oxidization of leucoanthocyanidin to form cyanidin,
pelargonidin, and delphinidin is catalyzed by anthocyanin
synthase (Figure 2;Cheng et al., 2014;Galland et al.,
2014). Anthocyanins, which are responsible for purple to blue
pigmentation, represent the bulk of the flavonoids present
in black and purple rice (Pereira-Caro et al., 2013;Zhang
et al., 2015). The compounds cyanidin-3-O-glucoside and
peonidin-3-O-glucoside are the most prominent, but also
represented are cyanidin-3,5-diglucoside, cyanidin-3-O-(600-O-p-
coumaroyl)glucoside, pelargonidin-3-O-glucoside, peonidin-3-
O-(600-O-p-coumaroyl)glucoside, and cyanidin-3-O-arabidoside.
Red and white rice grains have been classified as lacking
anthocyanin (Gunaratne et al., 2013;Xiongsiyee et al., 2018),
but both Boue et al. (2016) and Ghasemzadeh et al. (2018)
have been able to detect a low level in both red and brown
rice accessions. Unstable anthocyanidins can be converted
into the colorless flavan-3-ols epiafzelechin, epicatechin, and
epigallocatechin through the action of anthocyanin reductase,
and when glycosylated, a wide array of distinct molecules are
generated (Ko et al., 2006;Sasaki et al., 2014;Kim et al., 2015;
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FIGURE 2 | Secondary metabolism in rice. (A) A schematic representation of the shikimic acid pathway. DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate;
DAHPS, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; DHQ/SDH, 3-dehydroquinate dehydratase/shikimate 5 dehydrogenase; DHQS,
3-dehydroquinate synthetase; DHS, 3-dehydroshikimic acid; SDH, shikimate dehydrogenase; SK, shikimate kinase; S3P, shikimic acid 3-phosphate; EPSPS,
5-enolpyruvylshikimate 3-phosphate synthase; EPSP, 5-enolpyruvylshikimate 3-phosphate; CS, chorismate synthase; CM, chorismate mutase; PAT, prephenate
aminotransferase; ADT, arogenate dehydratase; PDT, prephenate dehydratase; PPY-AT, phenylpyruvate aminotransferase (Tzin and Galili, 2010;Widhalm and
Dudareva, 2015;Santos-Sánchez et al., 2019). (B) Possible routes to the production of benzoic acid, benzoic acid-derived compounds and lignin. CNL,
cinnamate-CoA ligase; CHD, cinnamoyl-CoA-dehydrogenase/hydratase; KAT1, 3-ketoacyl-CoA thiolase; TE, CoA thioesterase; BA2H, benzoic acid 2-hydroxylase;
BALDH, benzaldehyde dehydrogenase; AO, aldehyde oxidase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; ICS, isochorismate synthase; CCR,
cinnamoyl-CoA reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; F5H, ferulate 5-hydroxylase; CSE, caffeoyl shikimate esterase; COMT, caffeic acid
O-methyltransferase; CAD, cinnamyl alcohol dehydrogenase; LAC, laccase; POD, peroxidase; p-HBD, p-hydroxybenzaldehyde; HBDS, 4-hydroxybenzaldehyde
synthase; HCHL, 4-hydroxycinnamoyl-CoA hydratase/lyase; HBD, 4-hydroxybenzaldehyde dehydrogenase (Qualley et al., 2012;Gallage and Møller, 2015;Widhalm
and Dudareva, 2015;Liu et al., 2018). (C) Flavonoid metabolism. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthetase;
CHI, chalcone isomerase; F30H, flavone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanin synthase; ANR, anthocyanin reductase; GT,
glucosyltransferase; LAR, leucoanthocyanidin reductase; MT, O-methyltransferase; F2H, flavanone 2-hydroxylase (Chen et al., 2013;Galland et al., 2014). The
square dot arrows indicates steps which have not yet been fully elucidated, while the black arrows indicate steps supported by genetic evidence.
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Figure 2). Although the major enzymes operating in the
flavonoid pathway are well known and their encoding genes have
been identified (Table 2), many aspects underlying the synthesis
of these pigments in rice have yet to be fully elucidated.
THE GENETIC BASIS OF RICE GRAIN
PIGMENTATION
The rice genome harbors at least two genes encoding chalcone
synthetase: CHS1 on chromosome 11 and CHS2 on chromosome
7 (Shih et al., 2008;Han et al., 2009;Cheng et al., 2014),
each contributing to flavanone biosynthesis. For the production
of proanthocyanidins, three flavone 3-hydroxylase are relevant:
namely F3H-1 (chromosome 4), F3H-2 (chromosome 10),
and F3H-3 (chromosome 4; Kim et al., 2008;Park et al.,
2016). Two anthocyanin synthases are critical for the synthesis
of anthocyanins, namely ANS1 (chromosome 1) and ANS2
(chromosome 6) (Shih et al., 2008;Table 2).
Rc Role in Red Pericarp in Ancestral Rice
White grained rice was selected during rice’s domestication.
The two complementary genes Rc (on chromosome 7), which
encodes a basic helix-loop-helix (bHLH) transcription factor,
and Rd (chromosome 1) encoding a form of dihydroflavonol
4-reductase, an enzyme which enhances the accumulation of
proanthocyanidin, are together responsible for the red pericarp
color. Rc is closely associated with shattering and grain dormancy
(Sweeney et al., 2006), so therefore was selected against during
domestication. Rc-Rd genotypes produce red grain, while Rc-
rd genotypes produce brown grain (Furukawa et al., 2006).
The three common Rc alleles are the wild type Rc, and
mutant alleles Rc-s and rc.Rc-s differs from Rc due to the
presence of a premature stop codon, while rc lacks a 14 bp
stretch of the wild type sequence (Furukawa et al., 2006;
Sweeney et al., 2006). Carriers of rc produce a colorless
pericarp, while those of Rc-s produce a range of pericarp
pigmentation (Sweeney et al., 2007). A number of variants
have been identified as restoring the wild type (red) pericarp
pigmentation: Rc-g carries a 1 bp deletion 20 bp upstream
of the 14 bp rc deletion (Brooks et al., 2008), while Rcr
features a 44 bp deletion upstream of the 14 bp segment,
which restores the wild type reading frame (Ferrari et al., 2015).
Most varieties of African domesticated rice (Oryza glaberrima)
produce a red pericarp, and white variants harbor a loss-of-
function mutation in Rc. An exception is the O. glaberrima
specific mutation rc-gl, which carries a premature stop codon
146 bp upstream of the site of the Rc-s point mutation
(Gross et al., 2010).
Regulatory Cascade Influencing Purple
Rice Color
Anthocyanins are responsible for the black-purple pigmentation
in rice grain. The variation seen in pigmentation intensity has
been taken to imply that the trait is under polygenic control,
involving as yet unidentified genes (Ham et al., 2015). A number
of publications report the identification of rice genes that
regulate anthocyanin production, each adopting a different gene
coding system, which only adds to the confusion. According
to Hu et al. (1996), two classes of regulatory gene (R/B and
C1/Pl) govern both the accumulation of anthocyanin and the
regulation of its deposition. Two Rgenes have been characterized:
Ra maps to chromosome 4 and Rb to chromosome 1. The
former gene is thought to be a homolog of the maize R/B
gene. Three alleles of Pl (chromosome 4) have been identified,
namely Plw,Pli, and Plj, and each is responsible for a distinctive
pattern of pigmentation. Plwactivates anthocyanin synthesis
in most of the aerial parts of the rice plant (although not
in either the stem or the internode). The Pl locus harbors
the two genes, OSB1 and OSB2, each of which encodes a
bHLH transcription factor (Table 2;Sakamoto et al., 2001).
Other studies found the purple pericarp trait to be genetically
determined by the dominant complementary genes Pb (synonym
Prp-b) and Pp (synonym Prp-a), mapping to chromosomes 4
and 1, respectively (Table 2;Rahman et al., 2013;Ham et al.,
2015). While the product of Pb appears to be responsible for
the accumulation of pigment in the pericarp of brown grain,
that of Pp increases the amount of the pigment, giving rise
to purple grain. The number of copies of the Pp gene present
is correlated with the intensity of the purple pigmentation
(Rahman et al., 2013). In the absence of Pp, plants harboring
Pb produce grain with a brown pericarp, while the pericarp of
Pp carriers, lacking Pb, are white (Rahman et al., 2013). The
Pb locus comprises of two genes, a myc transcription factor
(Ra), along with bHLH16. The bHLH16 has been shown to be
involved in proanthocyanidin synthesis, while Ra is involved
in anthocyanin synthesis. Ra and OSB1 are believed to have
synonymous functions (Hu et al., 1996;Sakamoto et al., 2001;
Caixia and Qingyao, 2007). A 2 bp (GT) insertion in exon
7 of Ra abolishes purple pigmentation (Caixia and Qingyao,
2007;Lim and Ha, 2013;Rahman et al., 2013). Similarly,
Sakulsingharoj et al. (2016) have found that a 2 bp (GT)
insertion in exon 7 of OSB1, which along with a 1 bp deletion
of a guanine nucleotide in exon 8, results in a threonine for
methionine substitution at position 64, resulting in a white grain
phenotype. Carriers of the three loci Kala1 (chromosome 1),
Kala3 (chromosome 3), and Kala4 (chromosome 4) express a
black pericarp trait (Maeda et al., 2014). It has been suggested
that Kala4 is synonymous with Pb, and Kala1 with Pp.Kala4
encodes a bHLH transcription factor and corresponds to OSB2
(Table 2). OSB2 regulates a number of genes encoding enzymes
involved in anthocyanin synthesis, including F3H,DFR, and
ANS (Sakulsingharoj et al., 2014). The chromosomal region
harboring Kala1 includes Rd (dihydroflavonol 4-reductase).
Kala3 is likely be a synonym of MYB3 (Maeda et al., 2014).
The black grain phenotype occurring in tropical japonica
germplasm has been attributed to structural variants in the
Kala4 promoter sequence. Oikawa et al. (2015) have proposed
that Kala4 has been introgressed several times from japonica to
indica germplasm.
The R2R3-MYB transcription factor Os06g0205100 has been
proposed as a candidate for the Cgene, functioning as a possible
activator of DFR and ANS (Saitoh et al., 2004;Rachasima et al.,
2017;Sun et al., 2018). Os01g0633500 (A1) is a dihydroflavonol
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TABLE 2 | Regulatory and structural genes shown to be involved in the biosynthesis of anthocyanin and proanthocyanidin in rice.
Locus Allelic
locus
Gene name Locus ID CHRXaPericarp References
Rice Annotation
Project (RAP)
Rice Genome Annotation
Project (MSU)
Regulatory genes
Kala1 Rd OsDFR Os01g0633500 LOC_Os01g44260 1 Red and black Furukawa et al., 2006;Shih
et al., 2008;Maeda et al.,
2014;Sun et al., 2018
Pp 1 Red and black Caixia and Qingyao, 2007
Kala3 OsMYB3 Os03t0410000 LOC_Os03g29614 3 Black Maeda et al., 2014
Kala4 PlwOSB1/Pb/Ra Os04g0557800 LOC_Os04g47080 4 Hu et al., 1996;Sakamoto
et al., 2001;Caixia and
Qingyao, 2007;
Sakulsingharoj et al., 2016
OSB2 Os04g0557500 LOC_Os04g47059 4 Sakamoto et al., 2001;
Sakulsingharoj et al., 2014;
Oikawa et al., 2015
Rc Rc-s bHLH Os07g0211500 LOC_Os07g11020 7 Red Sweeney et al., 2006
Rc 7 Red Furukawa et al., 2006
rc 7 White Furukawa et al., 2006;
Sweeney et al., 2006
Rc-g 7 Red Brooks et al., 2008
Rcr7 Red Ferrari et al., 2015
Rc-gl 7 White Gross et al., 2010
Chromogen OsC1 Os06g0205100 LOC_Os06g10350 6 Black Saitoh et al., 2004;
Rachasima et al., 2017;
Sun et al., 2018
Structural genes
Chalcone synthetase (CHS) OsCHS1 Os11g0530600 LOC_Os11g32650 11 Common intermediate Shih et al., 2008
OsCHS2 Os07g0214900 LOC_Os07g11440 7 Common intermediate
Chalcone isomerase (CHI) OsCHI Os03g0819600 LOC_Os03g60509 3 Common intermediate Shih et al., 2008
Flavanone 3-hydroxylase (F3H) OsF3H-1 Os04g0662600 LOC_Os04g56700 4 Common intermediate Kim et al., 2008
OsF3H-2 Os10g0536400 LOC_Os10g39140 10
OsF3H-3 Os04g0667200 LOC_Os04g57160 4
Flavanone 30-hydroxylase (F30H) OsF30HOs10g0320100 LOC_Os10g17260 10 Common intermediate Shih et al., 2008
Leucoanthocyanidin reductase (LAR) OsLAR Os03g0259400 LOC_Os03g15360 3 Black rice Kim et al., 2015
Anthocyanidin synthase (ANS) OsANS1 Os01g0372500 LOC_Os01g27490 1 Black rice Shih et al., 2008
OsANS2 Os06g0626700 LOC_Os06g42130 6
UDP-glycosyltransferase (UF3GT) OsUGT Os06g0192100 LOC_Os06g09240 6 Black rice Yoshimura et al., 2012
Os07g0148200 LOC_Os07g05420 7 Black rice
Anthocyanin reductase (ANR) OsANR Os04g0630800 LOC_Os04g53850 4 Black rice Kim et al., 2015
aCHRX: chromosome.
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reductase gene involved in anthocyanin synthesis (Table 2).
Thus, A1 and C1 determine the purple color of grain. The S1
gene (Os04g0557500) encodes a bHLH protein, and contributes
to hull-specific pigmentation. The presence of a functional
copy of both C1 and S1 has been shown to be required for
hull pigmentation, while the product of A1 acts as a catalyst
for the development of purple hulls (Sun et al., 2018). The
pattern of anthocyanin pigmentation is determined by the
allelic status of A1,C1, and S1 (Sun et al., 2018). Several
authors have attempted to correlate sequence variants of a
number of regulatory genes, e.g., C1 and OSB2, with phenotypic
variation in rice grain pigmentation (Sakulsingharoj et al.,
2016;Rachasima et al., 2017;Sun et al., 2018). Lachagari
et al. (2019) conducted comparative genomics in 108 rice lines
and identified novel allelic variants in a number of genes
belonging to the flavonoid pathway, cytokinins glucoside, and
betanidin degradation biosynthesis that were associated with
purple pigmentation. Although a number of genes responsible for
grain pigmentation have already been identified (Table 2), there
is still a possibility that additional genes and variants thereof,
remain to be discovered.
The Genetic Basis of Grain Pigmentation
Inferred From Quantitative Trait Loci
Analysis or Genome Wide Association
Studies
A number of attempts have been made to exploit the quantitative
trait loci (QTL) mapping approach as a means of inferring
the genetic basis of grain pigmentation (Table 3). Tan et al.
(2001) identified nine QTL in an analysis of flour pigmentation
in a recombinant inbred line (RIL) population. Three QTL
reflected variation in the CIE 1976 color parameter L(lightness),
two in a(red-green), and four in b(yellow-blue). In a
backcross RIL population, made from a cross between the
rice varieties “Kasalath” (red pericarp) and “Koshihikari” (white
pericarp), Dong et al. (2008) identified four QTL underlying
variation in red pigmentation, with the two largest effect
QTL co-locating with Rc and Rd, the two minor effect QTL
being novel. An analysis carried out by Matsuda et al. (2012)
suggested that flavonoid content was governed by genetic factors
which control flavone glycosylation. In a recent study 21
QTL, responsible for variation in the content and composition
of anthocyanin and proanthocyanidin, were identified (Xu
et al., 2017). While some mapped to locations occupied by
already known genes, others mapped to genomic regions
not previously identified as harboring genes involved in rice
grain pigmentation.
The genome wide association study (GWAS) approach,
which has certain advantages over QTL mapping (Korte and
Farlow, 2013), has been applied in a few cases to determine
the genetic basis of grain pigmentation (Table 3). Shao et al.
(2011) used GWAS to identify 25 marker–trait associations for
grain pigmentation: some related to pigment intensity, others
to hue angle, L, a, or b. Their analysis confirmed the
importance of Ra and Rc.Butardo et al. (2017) used GWAS
to uncover a number of single nucleotide polymorphism loci
(SNPs) linked to Rc. The 763 SNPs associated with pericarp
pigmentation uncovered by Yang et al. (2018) mapped to 6 of
the 12 rice chromosomes (chromosomes 1, 3, 4, 8, 10, and 11);
some of the most significantly associated SNPs lying close to
previously identified structural or regulatory genes, but others
map to regions not previously associated with variation in rice
grain pigmentation.
‘Omics Approaches Taken to Unraveling
the Mechanistic Basis of Grain
Pigmentation
High throughput genomics, including transcriptomics,
proteomics, and metabolomics, have contributed to the
unraveling of biochemical pathways underlying target traits.
By combining genetics with systems biology tools the target
genomic regions were narrowed down to identify candidate
genes and proteins influencing key nutritional traits of interest in
rice (Butardo et al., 2017;Anacleto et al., 2019).
Differential transcriptomic analyses between pigmented and
non-pigmented rice grains identified regulators and downstream
targets of flavonoid pathway genes (Oh et al., 2018). The high
anthocyanin content of black rice was associated with enhanced
transcription of genes encoding anthocyanidin synthase, while
high proanthocyanin content, characteristic of red rice, was
accompanied by a notable abundance of transcript for a gene
encoding leucoanthocyanidin reductase (Chen et al., 2013).
Transcript abundance of genes encoding chalcone synthetase,
chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol
4-reductase, and anthocyanin synthetase was compared in white,
black, and red rice grain by Lim and Ha (2013). Four genes
were markedly up-regulated in pigmented grain varieties, while
the gene encoding chalcone isomerase displayed a similar level
of transcription in both white and pigmented varieties. The
enhanced abundance of transcripts of chalcone synthetase,
flavanone 3-hydroxylase, and anthocyanin synthetase seen
in some black varieties implied a strong correlation between
transcription and pigment content. Sun et al. (2018) found
that flavonoid pathway genes were regulated by ternary MYB-
bHLH-WD40 transcriptional complexes (Xu et al., 2013;Zhang
et al., 2018). A microarray-based comparison of black and
white rice identified nearly 1,300 differentially transcribed
genes, of which 137 were predicted to encode transcription
factors belonging to 1 of 10 different classes (Kim et al.,
2011). When Kim et al. (2018) applied RNA-seq to analyze
differential transcription, it was concluded that the B-box protein
encoded by BBX14 was a key regulator of the anthocyanin
synthesis pathway. Anthocyanin production in pigmented
grain appeared to be induced and fine-tuned by BBX14 in
conjunction with the basic leucine zipper transcription factor
HY5. Both irradiation at a high light intensity and the plant’s
sugar content can influence anthocyanin and proanthocyanin
synthesis (Xu et al., 2013;Ma et al., 2018;Zhang et al.,
2018). Therefore it would be of value to search for linkages
between photoreceptor and light signal transduction elements
associated with anthocyanin/proanthocyanidin synthesis in
pigmented rice (Teng et al., 2005). While transcriptomic
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TABLE 3 | Quantitative trait loci identified for colored related traits, anthocyanin and proanthocyanidin.
No. Population Size Markers Trait category QTLs/QTNs Closest structural and/or
regulatory genes
Chromosome Effect (%) References
1 RILs 238 162 RFLP and 48
SSRs
Flour color 9 1, 3, 4, 5,6, 7, 8 4.3–25.4 Tan et al., 2001
L* (3) 5,6, 8 4.5–15.7
a* (2) 4, 7 6.9–10.5
b* (4) 1, 3, 6, 8 4.3–25.4
2 BRILs 182 162 RFLP Degree of red
coloration
4 1, 7, 9, 11 2.1–83.7 Dong et al., 2008
qDRC-1* Rd 1 3.6–3.7
qDRC-7* Rc 7 75.9 -83.7
qDRC-9 9 2.1–3.2
qDRC-11 11 3.3–3.4
3 RILs 182 126 SSRs Anthocyanin and
proanthocyanidin
21 1, 2, 3, 7, 8, 10, 12 3.8–34.8 Xu et al., 2017
ANC (8) 1, 2, 3, 7, 10 8.8–34.8
PAC (13) 1, 2, 7, 8, 10, 12 3.8–17.0
4 Diversity panel 416 100 SSRs and 10
gene markers
Grain color 25 1, 4, 6, 7, 8, 9, 10, 11, 12 1.39–86.68 Shao et al., 2011
L* (3) Ra,Rc 4, 7, 10 4.96–31.23
a* (8) Ra,Rc 1,4, 7, 8, 9, 11, 12 1.51–19.65
b* (6) Ra 4, 6, 8, 9, 10 4.38–49.82
c (3) Ra 4, 8, 10 1.39–3.99
H(5) Ra 4, 6, 8, 9 5.4–86.68
Phenolic and
flavonoid content
10 4, 7, 8, 9, 10 2.64–39.67
PC (4) Ra,Rc 4, 7, 8, 9 5.87–39.67
FC (6) Ra,Rc 4, 7, 8, 9, 10 2.64–35.35
5 Diversity panel 203 sequencing data Pericarp color (PC) 4 7, 10 Wang et al., 2016
Rc-s Rc-s 7
qPc10 F3H 10
6 Diversity panel 244 122,785 SNPs Red seed color snp_07_6067391 bHLH Butardo et al., 2017
7 Diversity panel 419 208,993 SNPs Pericarp
color_whole panel
763 1,3, 4, 7, 8, 10, 11 Yang et al., 2018
Rd 1
MYB family transcription
factors
10, 11
(Continued)
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Mbanjo et al. Colored Rice Nutritional Benefits
TABLE 3 | Continued
No. Population Size Markers Trait category QTLs/QTNs Closest structural and/or
regulatory genes
Chromosome Effect (%) References
WD domain, G-beta repeat
domain containing protein
8
Pericarp
color_indica
99
MYB family transcription
factors
10, 11
OsCHI 3
Kala4 4
Rc 7
WD domain, G-beta repeat
domain containing protein
8
OsCHI 3
Kala4 4
Rc 7
RILs, recombination inbred lines; BRILs, backcross-recombinant inbred lines.
analyses have succeeded in shedding some light on the
transcriptional regulation of secondary metabolites, unraveling
post-transcriptional and post-translation processes may well
provide further insights into the identity of the rate-limiting
steps of grain pigmentation (Merchante et al., 2017;Spoel,
2018).
The abundance of a given transcript and its translation
product are not always linearly related, due to post-
transcriptional regulation, translation and post-translational
processing, and peptide modification (Chen et al., 2017;Zhang
et al., 2017). Thus, a proteomic analysis can give a more
nuanced picture of the differences between pigmented and
non-pigmented grains, than is possible from a transcriptomic
analysis. A total of 230 differentially abundant proteins,
involved in various metabolic processes, were identified by
Chen et al. (2016) from a comparison between black and
white grains sampled at five stages of grain development.
A number of proteins involved in the synthesis of flavonoids
and sugars were found to be more abundant in the black
grain, while proteins associated with signal transduction,
redox homeostasis, photosynthesis, nitrogen metabolism, and
tocopherol synthesis were less abundant. In particular, chalcone
synthetase was pinpointed as a key component required for the
synthesis of anthocyanin.
Metabolomic analyses have also been successfully used
to characterize the cellular composition of rice (Table 1).
Comparative metabolome studies of black, red, and non-
colored rice revealed that various anthocyanins, tocopherol,
fatty acid methyl esters, free sugars, and fatty acids were
found to be significantly different (Frank et al., 2012). de
Guzman et al. (2017) were able to monitor more than 1,000
metabolites in a screen of several rice varieties differing with
respect to their nutritional quality and glycemic response (de
Guzman et al., 2017). Comparisons of the grain metabolomes
of diverse rice accessions have revealed that a substantial
degree of variation exists at this level (Gong et al., 2013;
Pereira-Caro et al., 2013). However, the grain metabolome is
highly dynamic, responding strongly to the plant’s external
environment, so this variation is as a consequence of genetic
and environmental variation and the interaction there-
of. Correlation analyses carried out between individual
metabolites have nevertheless revealed the regulation of the
grain metabolome, with clusters of co-accumulated metabolites
appearing to be under the control of shared genetic factors
(Matsuda et al., 2012, 2015).
The application of ‘omics-based platforms have begun to
reveal the genetic and biochemical basis of the grain pigment
parameters a, b, L, hue, and chroma. While transcriptomic
and metabolomic analyses have identified certain important
structural and regulatory genes influencing core components of
flavonoid synthesis (Lee et al., 2015;Oh et al., 2018), what is
still lacking is a comprehensive understanding of the molecular
machinery underlying key metabolic processes such as the
polymerization and transport of tannins. A more integrated
approach, focusing on identifying linkages between regulatory
networks is needed (Figure 2). Combining diverse datasets
facilitates the reconstruction of regulatory networks and the
Frontiers in Genetics | www.frontiersin.org 13 March 2020 | Volume 11 | Article 229
fgene-11-00229 March 12, 2020 Time: 18:56 # 14
Mbanjo et al. Colored Rice Nutritional Benefits
identification of key modulators (Butardo et al., 2017;Wambugu
et al., 2018). As an example, an exploration of the key genetic
influences affecting grain amylose–amylopectin composition
has implicated two genomic regions, one on chromosome 6
and the other on chromosome 7 (Butardo et al., 2017). The
genetic region on chromosome 7 is in the vicinity of Rc and
includes a haplotype associated with increased amylose and
reduced accumulation of short chain amylopectin. The bHLH
transcription factor encoded by Rc activates the gene encoding
dihydroflavonol 4-reductase, thereby influencing the formation
of red pigmentation. The same transcription factor has also
been proposed to act as a regulator of starch structure, as it
engages within the network regulating granule-bound starch
synthase activity.
Conserving and Utilizing Pigmented Rice
Landraces for Future Breeding
Although rice landraces with pigmented grain represent
an important genetic reservoir for rice improvement, these
populations are rapidly being lost as a result of the introduction
of more productive, modern white rice varieties. Some of
these materials have been safeguarded in ex situ gene banks,
such as the major gene bank curated by the International
Rice Research Institute2. Few of these landraces have been
systematically characterized in terms of their grain end-
use quality, their nutritional features and potential health
benefits. Therefore, there is an urgent need to validate the
traditional knowledge associated with these materials with
scientific-based analyses. While the productivity of the landrace
materials is undoubtedly lower than that of modern white rice
varieties, their market value is potentially quite high, given
the growing consumer preference for nutritious foods (Islam
et al., 2018). Looking forward, there is a major opportunity for
breeding programs to develop productive pigmented varieties
(Voss-Fels et al., 2019).
Understanding the mode of inheritance of grain pigmentation,
identifying beneficial alleles of the key genes underlying these
traits, and developing trait-specific markers, will contribute
to accelerating efforts to breed high yielding pigmented rice
varieties. Advanced generation breeding lines of pigmented
lines have been developed (Bhuiyan et al., 2011;Arbelaez
et al., 2015). A black rice line has been developed in the
genetic background of a leading Japanese white rice variety
(Koshihikari); which has eating quality superior to that of the
widely cultivated black rice variety “Okunomurasaki” (Maeda
et al., 2014). Crosses have been initiated between pigmented
and non-pigmented varieties to develop pigmented varieties
adapted to the growing conditions in Kazakhstan (Rysbekova
et al., 2017). The Thai aromatic, deep purple indica-type rice
variety “Riceberry” has developed a reputation for its health-
promoting properties. Riceberry combines the desirable features
of two prominent rice varieties, one a local, non-glutinous
purple rice and the other an aromatic white jasmine rice
(Waiyawuththanapoom et al., 2015;Gene Discovery Rice and
Rice Science Center, 2017). Two improved pigmented varieties
2www.irri.org/international-rice-genebank
(the red rice “Rubi” and the black rice “Onix”) have been released
in Brazil (Wickert et al., 2014).
CONCLUSION
Pigmented rice varieties are gaining popularity among
consumers, and demand is only expected to rise. The seed
supply chain of pigmented rice is weak and thus rice value chain
opportunities have to evolve to meet the current nutritional
demand. Production of pigmented rice using landraces is
unable to meet market demand, emphasizing the need to
genetically improve these landrace materials. Systematic
nutritional characterization of the 130,657 accessions curated
by International Rice Research Institute’s gene bank and
Africa Rice3, which including pigmented entries, will create
new avenues for nutritional diversification that reaches lower
income target countries. These, as well as other, national ex
situ collections, represent a valuable source of genetic variation
for the improvement of pigmented rice, providing materials
to elucidate the genetic basis of grain pigmentation and
associated nutrition-related traits. The process of identifying
as yet unknown genes influencing flavonoid metabolism
and grain pigmentation could be accelerated by whole
genome re-sequencing, allowing novel allelic variants to be
harnessed for use as markers. Fine mapped genetic regions
associated with proanthocyanidins and anthocyanin needs
to be undertaken to develop quality markers to support
marker-assisted-selection breeding of these nutritional traits
into high yielding rice backgrounds. A systems approach to
study implication of diet based health benefits would require
holistic understanding of the molecular basis of human
health benefits of consuming grain pigmentation, enabling
the identification of the modulators involved to overcome the
prevailing double burden malnutrition and communicable
diseases in the target communities. While several health benefits
were shown to possess to consume pigmented rice, its texture
and palatability is found to be poor and thus its acceptance
rate is lower. To address this limitation, we need to explore
the genetic variation for the retention of flavonoids in the
milled endosperm.
AUTHOR CONTRIBUTIONS
EM and NS drafted the manuscript. TK, HJ, NE, CB, and LB
edited the part of the sections.
FUNDING
The authors thank the UK Biotechnology and Biological Sciences
Research Council’s Newton Fund Sustainable Rice Research
Initiative (Project BB/N013603/1) for its financial support.
3www.genebanks.org/genebanks/africarice/
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Mbanjo et al. Colored Rice Nutritional Benefits
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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Sreenivasulu. This is an open-access article distributed under the terms of
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... White rice makes a major contribution to the calorific intake of Asian and African populations, but its nutritional quality is poor compared to that of pigmented (black, purple, red orange, or brown) variants (5). The compounds responsible for these color variations are the flavonoids anthocyanin and proanthocyanidin, which are known to have nutritional value (162). The rapid progress made in the technologies underlying genome sequencing, the analysis of gene expression and the acquisition of global 'omics data, genetics of grain pigmentation has created novel opportunities for applying molecular breeding techniques to improve the nutritional value and productivity of pigmented rice 162). ...
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... Further, significantly high health benefits have been observed in pigmented rice varieties compared to white rice varieties of Sri Lanka (Gunaratne et al., 2013;Premakumara et al., 2013;Abeysekera et al., 2017aAbeysekera et al., , 2017bAbeysekera et al., 2020). At international level research studies, similar results have been observed by many researchers (Saikia et al., 2012;Irakli et al., 2016;Samyor et al., 2016;Meera et al., 2019;Mbanjo et al., 2020). However, in terms of healthy fatty acid profiles in the present study, we observed nutritionally sound properties in both traditional and new improved rice varieties as well as in both red and white rice varieties of Sri Lanka. ...
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In recent years, the health benefits of the pigmented rice varieties have been reported due to the richness of their bioactive compounds. Therefore, this study evaluated the antioxidant, total flavonoid, total phenolic, anthocyanin content, amino acid and individual phenolic compound quantification of nine Korean-grown rice varieties using spectrophotometric, HPLC-FLD-MS/MS and UHPLC Q-TOF-MS/MS methods. Our research found that the free fractions of DM29 (red rice) had the highest free radical scavenging ability of ABTS and DPPH. In contrast, the highest ferric reducing antioxidant power was observed in the 01708 brown rice variety. The majority of phenolic compounds such as quercetin, ferulic acid, p-coumaric acid, ascorbic acid, caffeic acid and genistein were found in the DM29 sample. The phenolic content of rice varies depending on its color, with DM29 red rice having the highest TPC, TFC and TAC levels. At the same time, the presence of the majority of amino acids was quantified in the 01708 and GR (Gangwon) brown rice varieties. According to this study, colored rice varieties are high in amino acids, phenolic compounds and antioxidants. This research would be beneficial in furthering our understanding of the nutritional value of different colors of rice and their high potential as a natural antioxidant. Keywords: antioxidants; anthocyanins; amino acids; phenolic phytochemicals; rice
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