Duo Xia,Hao Zhou,Yipei Wang,Pingbo Li,Pei Fu,Bian Wu,Yuqing He*

National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070, Hubei,China

Keywords:Anthocyanins Flavonoid Biosynthesis Genetic basis Black rice Oryza sativa L.

ABSTRACT Anthocyanins are a major subclass of flavonoids that have diverse biological functions and benefit human health.In rice(Oryza sativa),the various colors shown by organs are due mainly to the accumulation of anthocyanins and are traits associated with domestication.Elucidating the genetic basis of anthocyanin biosynthesis in rice would support the engineering of anthocyanins as well as shedding light on the evolutionary history of O.sativa.We summarize recent progress in rice anthocyanin biosynthesis research,including gene cloning,biosynthetic pathway discovery,and study of the domestication process.We discuss the application of anthocyanin biosynthesis genes in rice breeding.Our object is to broaden knowledge of the genetic basis of anthocyanin biosynthesis in rice and support the breeding of novel rice cultivars.

1.Introduction

Plant coloration is determined mainly by three types of plant pigments besides chlorophyll:anthocyanins,betalains,and carotenoids,which function in photosynthesis,defense,and reproduction[1].Anthocyanins,a major subclass of flavonoids,are the main secondary metabolites that give color to rice tissues[2].Anthocyanin accumulation in plants has diverse functions,including resisting UV radiation,participating in hormone regulation,and responding to biotic and abiotic stress,and is beneficial to human health[3-6].In rice,anthocyanins color tissues and give them various functions(Fig.1).Anthocyanin accumulation in the stigma and apiculus is a recognizable characteristic in cultivated rice[7,8].Rice cultivars with purple leaves can be planted as guard rows to separate different materials.Black rice cultivars have commercial value for the antioxidant,health-promoting activity of rice pericarp anthocyanins [9-11].Rice anthocyanin accumulation is a domestication-associated trait that is useful for tracing the evolutionary history of rice.

Since Mendel’s experiments in the 19th century,the potential of plant pigmentation for revealing basic rules of genetics and biochemistry has been recognized.The visual evidence of pigments accumulating in tissues has led to uncovering the molecular basis of anthocyanin biosynthesis in model plants such as petunia and maize[12].Flowers and crops with diverse colors have also been bred based on these achievements.With the rapid development of functional and comparative genomics,knowledge of the anthocyanin biosynthetic pathway in rice has made advances.This review summarizes these advances,with the aim of throwing light upon the evolutionary history of rice cultivation and assisting in the breeding of rice cultivars accumulating various anthocyanins.

2.Anthocyanin biosynthetic pathway

Anthocyanins are water-soluble secondary metabolites,classified into three categories:pelargonidin,cyanidin,and delphinidin[13].The biosynthesis of anthocyanidins occurs in a branch of the flavonoid synthetic pathway,starting with malonyl-CoA and p-coumaroyl-CoA(Fig.2)[3,5,14,15].Under catalysis by chalcone synthase(CHS),three acetate units from malonyl-CoA combine with p-coumaroyl-CoA to yield tetrahydroxychalcone,the basic skeleton of anthocyanins.The yellow-colored tetrahydroxychalcone is converted to colorless naringenin under catalysis by chalcone isomerase (CHI) and is further converted to dihydrokaempferol(DHK)by flavanone 3-hydroxylase(F3H).DHK can subsequently produce dihydroquercetin(DHQ)and dihydromyricetin(DHM)via hydroxylation by flavonoid 3′-hydroxylase(F3′H)and flavonoid 3′5′-hydrolase(F3′5′H),respectively.Next,the three colorless dihydroflavonols(DHK,DHQ,and DHM)are converted to anthocyanins in three steps.The first step is reduction of dihydroflavonols by dihydroflavonol reductase(DFR)to produceleucoanthocyanidins.These are converted sequentially to anthocyanidin 3-O-glucoside via oxidation by leucoanthocyanidin oxidase(LDOX)and glycosylation by 3-glucosyl transferase(3GT).Anthocyanidin 3-O-glucosides can be further glycosylated,methylated,and acylated to produce decorated anthocyanins with various colors.Plants produce and accumulate various anthocyanidin 3-O-glucosides.In colored rice,the most abundant being cyanidin 3-O-glucoside[16,17].The red color of rice seeds results from the accumulation of proanthocyanins,another subclass of flavonoid[3].Proanthocyanin synthesis starts with the reduction of leucoanthocyanidin or cyanidin via catalysis by leucoanthocyanidin reductase(LAR)or anthocyanidin reductase(ANR),respectively,and the products are transported to the vacuole to produce brown proanthocyanin derivatives[18].

Fig.1.Genetic diversity of rice color.(a)Rice pericarp color diversity from white,light red,red,and brown to black.(b)Rice seed color diversity.Left side shows seed morphology and right side the corresponding grain morphology.(c-f)Leaf(c),apiculus(d),stigma(e),sheath and stem(f),and plant(g)color diversity.

Fig.2.Simplified diagram of the anthocyanin biosynthetic pathway[3,4].Words in red represent synthesis enzymes and those in green represent the functional genes that encode the corresponding enzymes.CHS,chalcone synthesis;CHI,chalcone isomerase;F3H,flavanone 3-hydroxylase;DHK,dihydrokaempferol;F3′H,flavonoid 3′-hydroxylase;F3′5′H,flavonoid 3′5′hydroxylase;DHQ,dihydroquercetin;DHM,dihydromyricetin;DFR,dihydroflavonol 4-reductase;LDOX,leucoanthocyanidin oxidase;3GT,3-glucosyl transferase;LAR,leucoanthocyanidin reductase;ANR,anthocyanidin reductase.The biosynthetic pathway follows Grotewold and Koes[14].

3.Anthocyanin-biosynthesis genes

The genes involved in anthocyanin biosynthesis have been well described and isolated in comparison with other metabolismassociated genes in plants.They can be classified into two categories:structural and regulatory genes(Table 1).Structural genes encode functional enzymes that catalyze anthocyaninbiosynthesis reactions and regulatory genes encode mainly transcription factors(TFs)that regulate the expression of structural genes[12].

3.1.Structural genes

The main enzymes that participate in the biosynthesis of anthocyanins are CHS,CHI,F3H,F3′H,F3′5′H,and DFR(Fig.2).As shown in Table 1,many plant structural genes encoding biosynthetic enzymes have been cloned and characterized.

CHS catalyzes the initial step of anthocyanin biosynthesis.There are 12 different CHS genes in petunia,of which only four(CHSA,CHSB,CHSG,and CHSJ)are expressed[19].WHP and C2,two CHS genes in maize,control CHS activity in pollen and seeds,respectively[20,21].The rice Os-CHS1 cDNA clone,first isolated from a leaf cDNA library of an indica cultivar,has extensive sequence identity with maize CHS genes[22].

CHI functions in the second step of anthocyanin biosynthesis,accelerating the isomerization of p-coumaroyl-CoA to produce naringenin.CHI-A and CHI-B,two CHI genes isolated from Petunia hybrida,are expressed in all floral organs and immature anthers respectively[23].The petunia Po gene,corresponding to CHI-A,controls CHI expression in anthers[24].The maize ZmCHI1,isolated by gene similarity,shows 55%and 58%identity to CHI-A and CHI-B in petunia respectively[25].The rice OsCHI gene is orthologous to ZmCHI1,and mutation of OsCHI led to a golden hull and internode phenotype[26,27].

F3H functions in producing the colorless precursors of anthocyanins.An3 is a functional gene of F3H in petunia,and F3H is the only isolated gene of F3H in maize[28,29].There are three F3H homologs in rice:OsF3H-1,OsF3H-2,and OsF3H-3,among which OsF3H-2 shows the highest expression level and OsF3H-1 the highest enzyme activity[30].LOC_Os03g03034 is another gene encoding F3H,playing a role in BPH resistance[31].

DFR catalyzes the conversion of dihydroflavonols into the corresponding leucoanthocyanidins.Among three DFR genes identified in petunia,only dfrA/An6 is transcribed in floral tissues[32].The A1 gene is a functional DFR gene in maize,and a recessive mutation at this locus leads to a colorless aleurone layer[33-35].The rice DFR gene,OsDFR/Rd,is identical to the A1 gene[36].

Several genes encoding F3′H,F3′5′H,ANS,and 3-GT have been cloned in maize:Pr1,A2,Bz1,and Bz2,respectively[37-40].OsAns is identical to the maize ANS-encoding gene A2,and its mutant lines N2BB and G922 accumulate detectable amounts of proanthocyanins and leucoanthocyanidins[2].Via the rice annotation project[41],several genes encoding F3′H,ANS,and UGT have been annotated in rice(Table 1).

3.2.Regulatory genes

Three main types of genes regulate anthocyanin biosynthesis in plants:MYB TFs,basic helix-loop-helix(bHLH)TFs,and WD40 transcriptional regulators.Major genes in these classes are listed in Table 1.

MYB TFs exert transcriptional control of multiple processes in higher plants[42,43].The R2R3 MYB TFs function in plant pigmentation[44].C1,the first cloned MYB type regulatory gene in maize,regulates the biosynthesis of anthocyanins in aleurone by regulating the transcription of Bz1 and A1[45-47].Pl,another copy of C1,functions mainly in the pigmentation of vegetative and floral organs in maize[48].An2,a main determinant of color differences in petunia,also encodes a MYB TF[49].As shown in Fig.3,there are many MYB TFs in the rice genome[50].However,only three genes have been reported to be involved in anthocyanin biosynthesis.The MYB TF OsC1 can bind to the promoter elements of OsDFRand OsAns and participates in the flavonoid pathway and stress response by regulating the expression of OsDFR and OsAns[4].The gene kala3 was reported[51]to encode a MYB TF,but its function has not yet been characterized.A purple leaf mutant revealed that mutation in OsPL,a MYB TF,affects anthocyanin biosynthesis and stress response[52].

Table 1Anthocyanin-biosynthesis genes.

Fig.3.Distribution of anthocyanin-associated genes on rice chromosomes.Yellow boxes represent regions in that rice genome containing MYB TFs.Red boxes indicate annotated bHLH TFs in the genome.Orange boxes indicate regions that contain MYB TFs and bHLH TFs.Pink circles indicate anthocyanin-biosynthesis genes that have been cloned.ANS1,ANS2,B1,B2,C1,CHS1,CHS2,CHI,F3H,F3H-1,F3H-2,F3H-3,F3′H,PAC1,P1,and UGT are gene abbreviations for OsANS1,OsANS2,OsB1,OsB2,OsC1,OsCHS1,OsCHS2,OsCHI,OsF3H,OsF3H-1,OsF3H-2,OsF3H-3,OsF3′H,OsPAC1,and OsP1.

bHLH TFs are widely distributed in plants and participate in many biological processes including signal transduction and hormone response[53-55].They also function in anthocyanin biosynthesis.The maize R and B genes,two bHLH domain-containing genes,act on the same anthocyanin-biosynthetic pathway[56,57].Lc and Sn are another two bHLH TFs in maize and can complement the petunia an2 and an11 mutant phenotype respectively,indicating that the regulatory genes controlling anthocyanin pigmentation are functionally conserved in plants[58-60].jaf13 and an1 were two bHLHs in petunia,and ectopic expression of an2 and jaf13 increased pigmentation in petunia[49,61,62].As shown in Fig.3,there are a total of 167 bHLHs in the rice genome,distributed over the 12 chromosomes[55,63].Ra and Rb,two R genes isolated from rice,are located on chromosome 4 and 1 respectively.The Ra locus consists of two genes,Ra1 and Ra2.Ra activates the anthocyanin pathway in maize,while Rb induced pigmentation in maize suspension cells[64,65].The Pl locus in rice,consisting of OsB1 and OsB2,has been shown identical to Ra and OsB1 is allelic to Ra1.Both OsB1 and OsB2 can induce the anthocyanin pathway in transient complementation experiments[66].Rc,another bHLH gene located on chromosome 7,is a determinant of proanthocyanin biosynthesis in rice pericarp[67].The pale aleurone color1(pac1)locus,encoding a WD40 repeat protein,is required for anthocyanin biosynthesis in the aleurone layer and scutellum in maize[68].However,the function of OsPAC1 is unknown[69].It has been hypothesized[18,70]that the MYB,bHLH,and WD40 regulators form a transcription complex and bind to the promoter of structural genes to activate the anthocyanin-biosynthesis pathway.

In rice,the anthocyanin-biosynthesis pathway is described[71]as a CAP regulation system,with C,A and P standing for chromogen,activator,and tissue-specific regulator,respectively.In this system,both A and P are regulatory genes,while C represents structural genes.Both the expressions of structural and regulatory genes are closely linked to anthocyanin content.The expression of OsCHS,OsF3H,OsDFR and OsAns is higher in leaves and seeds of anthocyanin-enriched than in those of anthocyanin-undetectable cultivars,and the expression of these genes is increased during seed maturation.Among these genes,OsDFR and OsAns have relatively high expression and are specific to anthocyanin biosynthesis[30].In non-pigmented rice lines,OsB2 is not expressed and a substitution in the N-terminal interacting domain is observed in the OsB1 transcript[72].Whole-genome sequencing along with transcriptomic sequencing in black rice plants identified several genes involved in anthocyanin biosynthesis,including ANS1,F3H,UGT,and DFR[73].

4.Functional characterization of anthocyanin-biosynthesis genes in rice organs

Most anthocyanin-associated genes in rice were first isolated by cDNA hybridization to genes of maize relatives.With rapid advances in genotype methods and functional genomics,a growing number of anthocyanin pigmentation genes have been positionally cloned and functionally analyzed in rice.The regulatory mechanism of pigmentation in rice leaves,hull,and pericarp is becoming increasingly clear(Fig.4a).

4.1.Leaf

Mutation in chlorophyll biosynthesis genes such as OsCAO1[74],YGL1[75],and VAL1[76]leads to leaves whose color is mostly green but will turn to yellow or pale green.Rice leaves will turn purple with anthocyanin accumulation(Fig.1c),which was reported[69]to be controlled by OsC1,Rb,and Rd/DFR.OsC1 was reported[77]to influence cyanidin 3-O-glucoside content in rice leaves.Genomic sequence analysis(GWAS)showed that five null mutations of OsC1 lead to the non-anthocyanin-pigmented leaves(non-AL)phenotype,whereas some cultivars with functional OsC1 alleles still exhibited the non-AL phenotype.A second GWAS analysis of anthocyanin in rice leaves using cultivars with functional OsC1 alleles identified Rb,a bHLH gene on chromosome 1.A 6.5-kb retrotransposon insertion into the 5′UTR of Rb decreases the expression level of Rb and leads to the non-AL phenotype.However,eight cultivars with functional OsC1 and Rb alleles still show a non-AL phenotype,owing to a rare null mutation in the second exon of Rd/DFR.

4.2.Hull

The rice hull color varies from purple,red,or brown to yellow(Fig.1b).A C-S-A gene system has been proposed[78]for the regulation of rice hull color,in which C1 encodes a R2R3 MYB transcription factor,S1 encodes a bHLH protein and functions in a tissue-specific manner,and A1 encodes DFR.C1 interacts with S1 and activates the expression of A1.In the presence of a functional A1 allele,a high level of cyanidin will be accumulated and the hull will turn purple,whereas loss of function of A1 will lead to a brown hull color and the accumulation of flavonoids such as hesperetin 5-O-glucoside,rutin,and delphinidin 3-O-rutinoside.OsC1,Kala4,and Rd are the responsive genes for C1,S1,and A1 respectively.C1 along with S1 can upregulate the expression level of the structural genes in anthocyanin biosynthesis,but neither C1 nor S1 alone can influence the expression level of these genes.These results indicate that functional C1 and S1 are essential for the biosynthesis of anthocyanins and functional A1 is the foundation of rice hull pigmentation.

Fig.4.Anthocyanin-biosynthesis regulation and rice evolutionary history.(a)anthocyanin-biosynthesis regulation in various organs.Filled circles represent MYB transcription factors.Yellow and pink filled circles represent MYB transcription factors encoded by OsC1 and the uncharacterized Kala3 respectively.Filled ellipses represent bHLH transcription factors.Light blue,orange and gray filled ellipses represent bHLH encoded by Rb,Rc and Kala4/OsB2 respectively.Filled rounded rectangle represents the functional anthocyanin biosynthetic protein.Purple rectangle represents the functional enzyme encoded by DFR.The double helix represents the promoter sequence of specified genes.The question marks in ellipse and rectangle represent uncharacterized bHLH and functional enzymes.(b)The evolutionary history of anthocyanin biosynthesis genes in rice.OsC1,Rc,Rb,kala4/OsB2,and Rd represent the ancestral alleles of these genes.c1,c2,c3,c4,and c5 are null mutated alleles of OsC1.rc and Rc-s are null mutated alleles of Rc,rd2 is the null mutated allele of Rd,rb is the null mutated allele of Rb,and Kala4 is a gain-of-function mutation of kala4/OsB2.The mutations corresponding to these alleles are listed in Table 2.

4.3.Pericarp

Rice pericarp color is diverse,ranging from white,brown,and red to black(Fig.1a).The black pericarp is formed by the accumulation of anthocyanins and the occurrence of the red pericarp is due to the accumulation of proanthocyanins,whose synthesis pathway is a branch of the anthocyanin pathway that shares some of its biosynthetic genes[2,15,79].Pericarp color was reported[80]to be controlled by Ra,Rc,and Rd.Previous studies[17,81,82]have shown that cyanidin 3-O-glucoside and peonidin 3-O-glucoside are the two primary anthocyanins in black rice pericarps.Several studies[80,83,84]showed that Ra/PURPLE PERICARP(Pb,Prpb,and Prpb2)was responsible for purple pericarp,whereas purple color was dominant to white color.Ra was a bHLH gene located on chromosome 4 and a 2-bp(GT)deletion in the seventh exon was associated with the purple-pericarp phenotype[85].Kala1,Kala3,and Kala4 were reported to be associated with black pericarp in a near-isogenic line derived from the black rice cultivars Hongxienuo and Koshihikari.Kala1 was presumed to be Rd on chromosome 1 and Kala3 was considered to encode a MYB TF.Functional Kala4 will produce either purple or brown pericarp together with Kala1 and Kala3 or in the absence of either Kala1 or Kala3[51].Further research[86]revealed that Kala4/OsB2 encoded a bHLH TF and a large fragment insertion into the promoter markedly increased the expression level of Kala4/OsB2,showing that the black or purple pericarp was a gain-of-function mutation.Rc and Rd have been proposed to be the genes responsible for red-pericarp formation in rice.Functional alleles of Rc and Rd together produce red pericarp,whereas a functional allele of Rc alone produces brown pericarp[80,87].Rc encodes a bHLH TF,and either a 14-bp deletion or a C-A mutation inducing a premature stop and a truncated protein lacking the bHLH domain is able to turn the red pericarp to white[67,88,89].Besides Rc,Rd and Ra/Kala4/OsB2,qPC10 which encodes an F3H was responsible for pericarp color in aus and OSCHI,and several genes encoding LDOX were identified[90,91]as being associated with rice pericarp color.

4.4.Other tissues

Anthocyanin accumulation in leaf sheath,stigma,and apiculus will produce purple rice tissues(Fig.1d-g).Variation in apiculus coloration is caused by diversity of the C locus[71].OsC1,a homolog of the maize C1 gene,is the functional gene underlying apiculus coloration variation.The colored apiculus C1 allele inT65 contains three exons and encodes a 232-amino-acid protein containing a MYB domain at the N terminal,while the colorless apiculus allele in IR 36 and 868 is caused by a 10-bp deletion on the third exon,which leads to a frame shift and finally a loss of function of the protein[92].The OsC1 allele co-segregated with the coloration of both the apiculus and the leaf sheath,suggesting that OsC1 may be responsible for the variation of leaf sheath and apiculus color[93-95].

5.Evolutionary history of anthocyanins in rice

Rice has been domesticated for thousands of years and now is grown worldwide.Studies[96-101]investigating the evolutionary history of rice have indicated that genes such as sh4 for shattering and prog1 for erect growth were elected during domestication.Grain-quality traits including color,size,and lipid composition are also domestication-associated traits[102-104].

Grain color has been a target during domestication,given that the color of most cultivated rice is white and that of most wild rice is red.The domestication of Rc and Rd accounts for the selection of rice pericarp color[67,89,105].Loss-of-function mutation of Rc produces white kernels.About 97%of white-pericarp cultivars carry a 14-bp deletion that results in a premature stop codon and a nonfunctional rc allele.The few remaining white-pericarp cultivars harbor a C-A point mutation in exon 7,which is a null mutation and designated as Rc-s.Haplotype analysis of Rc revealed[89]that the 14-bp deletion arose from Geng and was introgressed into Xian and aus,and the C-A mutation originated in the aus subpopulation and was not widely disseminated during domestication.Wang[106]thought that the 14-bp deletion was selected in Xian before introgression from the Geng genepool.A unique null mutation in exon 7 of Rc inducing the formation of white pericarp in O.glaberrima was identified[107].Yet neither the 14-bp deletion nor the Rc-s mutation was observed in O.glaberrima.The mutation at the white O.glaberrima Rc locus was in the same region as the Rc-s mutation,suggesting that human selection was identical in separate domesticated species.But it has been suggested that the domestication process of white pericarp was different in aus from that in Geng and Xian(Fig.4b).Wang[90]reported that the Rc-s mutation turned the red pericarp to light red pericarp and the qPC10 mutation in the Rc-s background introduced white pericarp.Both Rc-s and qPC10 were selected in aus.Two functional SNPs of Rd:a G-T mutation in exon 1 named rd1 and a C-A mutation in exon 2 named rd2,are present in Geng.It was suggested[105]that the rd mutation occurred twice independently during Geng rice domestication(Fig.4b).The rd2 mutation was associated with the qsh1 mutation by genetic linkage.The rd1 mutation may have originated in the Philippines and been transferred to the ancestors of Japanese upland rice.The reason for the selection of rd was speculated[105,108]to be its linkage with the‘‘Green Revolution gene”sd1.

Unlike the red pericarp,the black rice trait is an acquired trait,which is not observed in any wild rice accessions(http://www.gramene.org/).In ancient China,black rice was called‘‘Emperor’s rice”or‘‘forbidden rice”for its rarity.Kala4/OsB2/Pb is reported[85,87,109]to be essential for purple-pericarp formation in rice.A large fragment insertion into the promoter of Kala4/OsB2 is a gain-of-function mutation that increases the expression of Kala4/OsB2 and was reported[86]to be the original genetic change that gave rise to black rice.The nucleotide diversity of Kala4/OsB2 in cultivated rice accessions revealed that the insertion first occurred in tropical japonica,and then spread to Xian and subsequently temperate japonica via continuous natural crossing and artificial selection of the black-rice trait(Fig.4b).

Most rice cultivars do not accumulate anthocyanins in leaves.This accumulation is controlled by OsC1,Rb,and Rd.Several mutations in OsC1 are observed in cultivated rice,and Xian and Geng carry loss-of-function mutations of independent origin.Thus,OsC1 allelic diversification might have resulted from mutations in the coding region rather than from recombination between preexisting alleles[92].OsC1 and Rb were reported[69]to have been selected independently.The OsC1 mutations c1 and c2 were selected in Xian and aus respectively,and the rb mutation was selected in Geng(Fig.4b).These negative selections of OsC1 and Rb led to the anthocyanin-non-accumulating leaf phenotype.The reason for this negative selection may be that anthocyanin pigmentation in rice leaves reduces the efficiency of photosynthesis,in turn reducing rice yield.

The origins of Xian and Geng are keys to the domestication of O.sativa[110].Several researchers[111-113]defend single-origin models of rice domestication and propose that Geng was first domesticated and that Xian was developed from crosses between Geng and wild rice.Others[106,114]support the model of independent domestication of Xian and Geng.Xian and Geng are speculated[103,109,115-117]to have originated from different wild rice accessions and undergone independent domestication processes.However,mutations in Rc,Rd,and Kala4/OsB2 originated in Geng and were introgressed into Xian,indicating that anthocyanin biosynthesis evolution is in accord with the single-origin domestication model.Given the complexity of the domestication process of O.sativa,it is still hard to establish conclusively the origin of cultivated rice.But the domestication process can be deduced from the evolutionary history recorded in the genome of cultivated rice[118].

6.Applications:black rice and genetic improvement

Black rice has become a delicacy and has drawn the attention of both consumer and breeders[9,80,86,119-127].Besides its high anthocyanin content,black rice contains high levels of amino acids,fatty acid methyl esters,and free fatty acids,giving it health advantages such as inhibition of cholesterol absorption,protection against angiogenesis diseases,and prevention of heart and cardiovascular diseases[120,128-130].Black rice is used as an ingredient in delicacies such as rice cakes,fried rice,porridge,and bread.Replacement of wheat flour with black rice flour in muffins improved its nutritional and antioxidant characteristics[131].Black rice porridge retained more active substances than cooked rice in a rice cooker and non-waxy black rice showed higher phenolic content and antioxidant capacities than waxy rice[132].Cooking methods influence the anthocyanin content in black rice,and cooking on a gas range led to lower loss of cyanidin-3-glucoside than pressure cooking or cooking in a rice cooker[133].Cooking non-waxy black rice on a gas range or as a porridge maximized its nutritional value.

Besides their health benefits,anthocyanins in rice have many other biological functions.The rice pericarp color has been reported[134,135]to be associated with seed dormancy and Rc was the functional gene responsible for seed dormancy.Rice anthocyanin biosynthesis is responsive to stresses such as drought,high salt,and ABA,and also responsive to disease resistance[136,137].It is feasible to increase resistance to biotic and abiotic stress and increase seed dormancy by increasing anthocyanin content in rice.

Increasing rice anthocyanin content is a target of transgenic breeding.Transforming the maize anthocyanin regulatory genes C1 and R and the structural gene C2 into a japonica variety,Tp309,led to anthocyanin accumulation in the leaves and leaf sheaths of transgenic plants and resistance to blast fungi Magnaporthe grisea[137].Increasing the expression of OsB2 increased anthocyanin accumulation in rice[138,139].Introducing the maize C1 and R-S genes driven by a rice endosperm specific promoter into a japonica cultivar resulted in high concentrations anthocyanins in the cells of four to five outer endosperm layers,but also in chalky endosperm[140].Overexpression of Lc in rice increased the anthocyanin content in floral organs:spikelets,anthers,and ovaries,but the pigmented flowers were sterile and unable to mature naturally[141].Overexpressing OsANS in a rice mutant NP increased the accumulation of a mixture of flavonoids and anthocyanins and increased antioxidant potential[142].

Pigmented rice cultivars have long been favored by breeders and consumers and their agronomic traits and quality have been improved[11].Maeda[51]developed a black rice introgression line by backcrossing Hong Xie Nuo with Koshihikari.The eating quality of the black rice NIL(near-isogenic lines),especially the overall value and glossiness,was superior to that of another black rice cultivar,Okunomurasaki.Breeding lines developed from crosses between white and black lines showed higher both phenolic content and anthocyanin content than the parental black rice,supporting the prospect of developing new rice cultivars with high antioxidant capacity and high yield[143].Chinese scientists and breeders have also made progress in black rice breeding.Pinhei 1,a black glutinous rice cultivar,could be used for producing functional rice products owing to its selenium-rich property[144].Zhongzi 4 is a nutrient-rich black rice cultivar bred by scientists from Chinese Academy of Agricultural Science.By introducing the thick-aleurone gene ta2,breeders increased the contents of total protein,lipids,minor elements,dietary fiber,and vitamins[145].Ziyunuo,whose color,aroma,and taste has reached the first-class standard of black glutinous rice,has become increasingly popular with consumers[146].

Despite the health benefits of black rice,anthocyanins are accumulated mainly in bran and will be removed when rice kernels are polished[124].Genetic engineering is a possible way to develop rice varieties with purple endosperm,as suggested by the breeding of‘‘golden rice”to increase the vitamin A content in rice[147-149].Flavonoids have been successfully synthesized at high levels in the endosperm via development of transgenic lines using endosperm-specific promoters to express genes involved in flavonoid biosynthesis[150].Zhu et al.[151]developed a novel biofortified germplasm,‘‘Purple-endosperm rice”,called Zijingmi in Chinese, using a high-efficiency vector system.Eight anthocyanin-associated genes driven by an endosperm-specific promoter are contained in this vector.Antioxidant activity was much higher in either unpolished or polished kernels of purpleendosperm rice lines than in transgenic-negative lines.Red rice accumulating proanthocyanin in the bran displayed more than sevenfold higher total antioxidant capacity and phenolic content than normal rice cultivars[123].A non-waxy red-pericarp mutant M-69 showed higher total phenolics,100-kernel weight,and grain yield than wild white-pericarp lines[80].A gene-editing approach using CRISPR/Cas9 restored the recessive rc allele by reverting the 14-bp deletion to an in-frame mutation,and three elite whitepericarp cultivars were successfully converted to red ones[152].

Most of the functional mutations regulating anthocyanin biosynthesis have been identified and several functional markers have been developed(Table 2).Rc and Rd are involved in red-pericarp formation,and OsB1,allelic to Ra1,is necessary for anthocyanin biosynthesis.Various alleles of these three genes are present in the genomic sequences of black,red,and white-grain cultivars.A C-A mutation in DFR/Rd is present in white-grain cultivars.Both black-pericarp and red-pericarp rice carry a C allele,and white-pericarp cultivars all carry the A allele,which leads to a premature stop codon.The Rc locus is non-functional in both black and white rice owing to a 14-bp deletion in the coding region that truncates the functional domain of the Rc protein.A GT insertion in OsB1,present in both red-rice and white-rice cultivars,causes a frameshift and a premature stop codon.Based on these findings,two CAPs(Cleaved amplified polymorphism sequence)markers and an INDEL marker(Table 2)have been developed to assist in molecular breeding of colored rice varieties[153].

7.Conclusions and future perspectives

Anthocyanins are the main metabolites that give rice vario us colors.Anthocyanin accumulation in rice tissues is controlled by three types of genes,MYB TFs,bHLH TFs,and a functional enzyme DFR(Fig.4).OsC1 is the only functionally characterized MYB TF in anthocyanin biosynthesis and is responsible for the coloration of leaf,anther,and hull.OsB2 is a bHLH TF that is essential forpurple-hull and black rice and Rc is another bHLH that accounts for red-pericarp formation.Rb is also a bHLH TF that is responsible for anthocyanin accumulation in rice leaves.Kala3 was proposed to be a MYB TF that is responsible for the black pericarp,but the function is unknown[154].

Several genes or QTL that regulate rice anthocyanin biosynthesis and coloration have been identified,many of which were genes that were not among the three types listed above.PSH1,a locus conferring purple leaf sheath,was mapped to a 23.5-kb region on chromosome 1,and six ORFs were annotated but none was reported to be involved in anthocyanin biosynthesis[155].IBF1/OSKF1 on chromosome 9,a gene encoding a kelch repeatcontaining F-box family domain,was reported to be a suppressor of brown pigmentation in rice hull furrows[156,157].An amino acid transporter Bh4 controls the black-hull phenotype in wild rice and a 22-bp deletion in the third exon disrupts the function of Bh4 and leads to the straw-white hull phenotype in cultivated rice[158].OsBBX14,a rice B-box protein,was reported to participate in rice grain anthocyanin biosynthesis.Two function-unknown genes,Os04g0577800 and Os04g0616400,were candidate genes for Plr4,a recessive locus for purple leaf[159].Many puzzles in rice anthocyanin accumulation and coloration await solution.

The evolutionary history of rice anthocyanin biosynthesis genes reveals that the purple-leaf trait was negatively selected and the black rice phenotype is a gain-of-function mutation.However,lower yield was observed for black rice than for white-pericarp lines with similar background[160].Rice hull has long been treated as a waste product,but studies[161,162]have demonstrated the antimutagenic and anticlastogenic potential of purple rice hulls.‘‘Green super rice”has become the goal of rice breeding since the beginning of this century and high yield with good quality is the main pursuit of rice breeders[163].With the improvement of living standards and health consciousness,increasing attention is paid to the quality and health benefit of food[164-166].With increasing knowledge of the pathways of anthocyanin biosynthesis and advances in molecular breeding,it is possible to breed novel rice cultivars that meet the demands for health,yield,and environmental compatibility at the same time.We expect that the time is coming when the genetic mysteries of rice color will be completely revealed,and we will be able to determine rice color as we wish.

CRediT authorship contribution statement

Duo Xia and Yuqing Heproposed the concept;Duo Xia,Hao Zhou,Yipei Wang,Pei Fu,Pingbo Li,and Bian Wudrafted the manuscript;Yuqing Herevised and finalized the manuscript.All the authors have participated sufficiently in the work to take public responsibility for all portions of the content.All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Program on R&D of Transgenic Plants(2016ZX08009003-004),the National Natural Science Foundation of China(91935303,32001530),the China Agriculture Research System(CARS-01-03),and the Postdoctoral Science Foundation of China(2020M682441).