Pericarp is the outermost layer of rice seed and located just beneath the hull of rice. All over the world, the rice varieties mostly possess white pericarp and few are red, brown, green, black or purple pericarp [9, 12, 17, 25]. Among them, black rice is characterized by dark purple pigments of anthocyanin accumulation in the pericarp [18, 22, 38].
Genetically, the pigmentation in pericarp of black rice requires two dominant complementary genes, PURPLE PERICARP A (Pp, Prpa and Prp1) and PURPLE PERICARP B (Pb, Prpb and Prp2) located on chromosomes 1 and 4, respectively [14, 28, 47]. The existence of two dominant genes Pb and Pp result purple color of rice pericarp. However, Pb in the absence of Pp produces brown pericarp and Pp alone produces ordinary white pericarp rice [13, 34, 45]. Additionally, the intensity of pericarp color in black rice also affected by the number of dominant Pp alleles in presence of dominant Pb alleles .
In black rice, high level of anthocyanins accumulation occurs during seed development stages result in a dark purple color pericarp [37, 41]. Anthocyanins are a group of natural pigments that belong to the family of flavonoids. Two major anthocyanin pigments namely cyanidin-3-O-glucoside (C3G) and peonidin-3-glucoside (P3G) are found in rice seed pericarp [22, 34]. Among them, C3G anthocyanin possesses biomedical properties including anti-oxidant, anti-cancer, and anti-inflammatory effects in human [15, 26, 29].
Although black rice has increased demand, the yield potentiality of colored rice is much lower as compared with white or hybrid rice [23, 31, 48]. The probable reasons for the low yield potentiality of black rice in comparison with white rice might be due to small sink size . We conducted a pilot project to observe the pigments deposition pattern during grain filling stage and its influence on yield. Therefore, it is urgent to explore the facts regarding the genetic makeup and deposition of anthocyanins in pericarp, and their relations with development of rice grain. In this review, we summarized the anthocyanin biosynthesis in pericarp, emphasizing inheritance pattern of the trait and the functions of their products on different physiological and agronomic traits including yield of black rice.
Anthocyanin pigments in pericarp of black rice
Black rice has a high level of purple pigments in pericarp layers , which results in dark purple color seed. This purple pigmentation in the black rice is the color of anthocyanin . Anthocyanins are a group of natural pigments that belong to the family of flavonoids which synthesized by a secondary metabolic pathway from the amino acid phenylalanine . Early studies reported that, cyanidin-3-O-glucoside (C3G) as a major anthocyanin in black rice, but the minor ones either malvidin-3-glucoside  or peonidin-3-glucoside (P3G) . Recently, four anthocyanins were identified in black rice namely cyanidin-3-O-glucoside, peonidin-3-glucoside, cyanidin-3-5-diglucoside, cyanidin-3-rutinoside (Fig. 1). A strong and identical peak of cyanidin-3-O-glucoside (kuromanin) was found in pericarp extract of purple pericarp rice, a trace amount of peonidin-3-glucoside was also found as well . In contrast, the accumulation of anthocyanin was not detected in pericarp extract of white and brown pericarp rice (Fig. 2). Cyanidin-3-O-glucoside as major pigment and peonidin-3-glucoside as minor pigment are also the core anthocyanins in seed pericarp of the grains such as purple wheat and purple rye .
Fig. 1.Chemical structures of most common anthocyanin in the pericarp of black rice [19, 42].
Fig. 2.Anthocyanin profile in progenies of black rice and white rice extract analyzed via HPLC. (A) HPLC profiles of the extracts from the dark-purple to white pericarp of rice seeds in F2 progenies. (B) Measurement of cyanidin-3-O-glucoside (C3G)in each pericarp of rice seeds. The high amount of C3G was extracted in dark purple seeds. However, relatively smaller amounts of C3G were extracted in the medium purple seeds of the F2 progenies. The brown and white seeds of F2 progenies contained no anthocyanin .
Genes involved in anthocyanin biosynthesis in black rice
Anthocyanins are derived from one molecule of p-coumaroyl-CoA and three molecule of malonyl-CoA which is originated by using phenylpropanoid and acetate pathway, respectively (Fig. 3). Several genes are involved in anthocyanin biosynthesis of rice and divided into two types; the structural genes (Table 1) are directly participating in the biosynthesis of anthocyanins, and the regulatory genes (Table 2) that control the expression of structural genes .
Fig. 3.The anthocyanin biosynthesis pathway in rice. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone3β-hydroxylase; F3’H, flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’, 5’-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, Anthocyanidin synthase; ANR, anthocyanidin reductase; LAR, leucoanthocyanidin reductase and UFGT, Flavonoid 3-0-glucosyltransferase[32, 37, 42].
Table 1.Structural genes involved in anthocyanin biosynthesis pathway in rice
Table 2.Regulatory genes involved in anthocyanin biosynthesis in rice seed pericarp
Structural genes for black rice
Anthocyanins with significant biological activities and a number of structural genes isolated from rice have been described in Table 1. The anthocyanin biosynthesis is catalyzed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid-3’-hydroxylase (F3’H), flavonoid-3’,5’-hydroxylase (F3’5’H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and flavonoid-3-O-glucosyltransferase (UFGT) [19, 21, 32, 42]. Generally, the first step in anthocyanin biosynthesis is condensation of three malonyl-CoA molecules and one p-coumaroyl-CoA molecule by the action of enzyme chalcone synthase (CHS) to produce a yellow naringenin chalcone. In rice (O. sativa), there are at least two genes CHS1 (LOC_Os11g32650) and CHS2 (LOC_Os07g11440) encoding CHS which are located on different chromosomes . After the action of chalcone isomerase (CHI), naringenin chalcone isomerized into a colorless naringenin flavanone stereospecifically and quickly. The naringenin flavanone is then hydroxylated by flavanone 3-β-hydroxylase (F3H) to produce an unpigmented dihydro-flavonol. In rice, three genes F3H1 (NM_001060692), F3H2 (AAL58118) and F3H3 (CAE02796) encoding F3H were identified and characterized . The dihydroflavonol is reduced to colorless leucoanthocyanidin by dihydroflavonol- 4-reductase (DFR). This leucoanthocyanidin is then converted into a colored anthocyanidins, catalyzed by anthocyanidin synthase (ANS). Two highly conserved ANS genes ANS1 (Os01g0372500) and ANS2 (Os06g0626700) located on different chromosomes were identified and characterized in rice . In the final step, anthocyanidins are glycosylated to form anthocyanins, catalyzed by the enzyme UDP glucose flavonoid-3-oxy–glucosyl transferase (UF3GT) (Fig. 3).
Over the last few years, many structural genes (Table 1) encoding the key enzymes involved in anthocyanin biosynthesis in rice have been cloned. Among them, two genes CHS  and CHI  were identified in rice based on sequence homology that involve in anthocyanin biosynthesis. A functional gene, DFR is responsible for red color in pericarp was established by over-expressing transgenic lines in rice . Similarly, transient assays showed that ANS gene of rice can complement the function of mutated a2 gene in maize kernels . Furthermore, biochemical roles of six genes of rice (CHS, CHI, F3H, F3’H, DFR and ANS) were well established by complementation assays in the Arabidopsis tt mutants .
Regulatory genes for anthocyanin of black rice
Regulatory genes (Table 2) influence the intensity and tissue specific pattern of anthocyanin accumulation and generally regulate expression of different structural genes of anthocyanin biosynthesis pathway at the transcriptional level . Two major classes of regulatory gene family, namely R/B family gene encode basic helix-loop-helix (bHLH) myc type protein  and C1/PI family gene encode the myb type protein  are known to regulate anthocyanin biosynthesis pathway in many plants. At least one gene from each family is required for anthocyanins pigmentation in tissue specific manner. Physical interactions between the R/B and C1/PI proteins are necessary to activate the expression of the key structural genes in anthocyanin pathway. However, the genes encoding a WD40 protein are also required for the expression of anthocyanin biosynthetic genes in maize seed . These are connected to form a complex in a hierarchical network to regulate the expression of the structural genes involved in the pathway .
Two basic genes namely, chromogen gene C and activator gene A are essential in the formation of anthocyanin pigments . The organ-specific regulatory gene P is necessary to confer tissue-specific accumulation of anthocyanin in plants . They are termed as localizer or distributor genes or regulatory genes of the pathway conditioning tissue specificity.
Rice seed pericarp becomes brown in color in presence of Pb alone (in absence of Pp), purple color in presence of both genes, and white color in absence of Pb (even in presence or absence of Pp) . Recently, three loci namely Kala1, Kala3 and Kala4 were identified to involve in pericarp pigmentation of black rice . It is also proposed that Kala1, Kala3 and Kala4 might correspond to C, A and P, respectively. Kala4 contains Pb gene that encodes a bHLH protein which is essential for color development in black rice pericarp . The Plw locus composed two highly homologous genes OSB1 and OSB2 encoding bHLH protein and confers pigmented leaf and pericarp of black rice . The Pb gene is probable to be the same gene OSB1 which is located in the Plw locus . Therefore, investigation of the Plw locus is still endured. Pp may be an allele of Kala1 as the genetic interaction of Kala4 and Kala1 is similar with that of Pb and Pp . However, Kala4 and Kala1 are urgent for anthocyanin biosynthesis in rice pericarp. Furthermore, Kala1 corresponds A gene which encodes the dihydrofavonol-4-reductase (DFR) enzyme and mapped on rice chromosome 1. DFR is a widely conserved gene which encodes an enzyme involved in both anthocyanin and pro-anthocyanin synthesis pathway . Kala3 might be a myb family transcription factor, act as a tissue-specific regulator and may be similar to P genes. Thus, Kala1 and Kala4 are necessary for anthocyanin biosynthesis and Kala3 might possess distinct seed pericarp tissue-specific transcription pattern . Molecular cloning has been conducted for the regulatory genes involved in anthocyanin biosynthesis in rice seed pericarp. The rice genes Ra1 and Rb , C1  and Pb [30, 34, 40, 44] have been cloned. Further, Pp gene cloning is required to complete understanding the genetic regulation of Prp trait.
Genetic analysis of purple pericarp traits in black rice
The inheritance of purple pericarp trait of rice is found to be dominant over white. The purple coloration in rice pericarp is determined by the complementary effect of two genes namely, Pp and Pb whose are located on chromosome 4 and 1, respectively [14, 47]. The Pp gene acts in a recessive epistasis fashion with the Pb gene for the production of purple pericarp in rice [13, 44]. However, the Prp trait has been defined as complementary interaction between Pb and Pp genes recently (Lee et al. 2016 under publication). The Pb gene was mapped on rice chromosome number 4 and suggested that, Ra gene may be similar to Pb gene, and purple pericarp characteristic of rice is caused by two bases deletion (GT) within exon 7 of gene Ra [34, 43]. The Pb gene alone is responsible for the pigments accumulation in the pericarp of brown colored seed was described in figure 5. Furthermore, purple pericarp rice requires the Pp gene to increase the content of the pigment from brown to purple (Fig. 5). We proposed that Pp gene regulate the anthocyanidin synthase which mediated the conversion of leucoanthocyanidin to anthocyanidin during anthocyanin biosynthetic pathway in rice. Therefore, Pb and Pp genes are involved in the purple pigmentation in rice pericarp, Pb- Pp- for purple pericarp, Pb- pppp for brown pericarp, and pbpb Pp-or pbpb pppp for white pericarp . Here, homozygous recessive allele of pb gene may be masked the expression of either allele of the Pp gene. Consequently, Pp itself does not produce any pigment in presence of pbpb. Therefore, the Pb and Pp genes involved in the purple pigmentation of the rice pericarp with epistatic gene interaction having segregation ratio of 9 purple: 3 brown: 4 white (Fig. 4). This gene interaction was also observed in inheritance of purple pericarp traits in wheat. It is recognized that two dominant complementary genes, Pp1 and Pp3, control purple pigments deposition in wheat pericarps .
Fig. 4.Segregation analysis of pericarp color of black rice. (A) Purple pericarp rice Oryza sativa L. japonica var. ‘Heugnambyeo’ was crossed with a white pericarp rice. F1 plants become purple color pericarp. Further selfing of F2 plants produced F3 seeds with purple pericarp, brown color pericarp, and white pericarp. (B) Phenotypes of the pericarp of cross materials. Purple pericarp rice was crossed with white-colored pericarp wild-type ‘Kumgangbyeo’ rice .
Fig. 5.Genetic analysis of crosses among black rice. (A) Genetic analysis of a cross between black rice PbPb PpPp and white rice pbpb pppp in their genotypes. (B) Genetic analysis of a cross between black rice PbPb PpPp and white rice pbpb PpPp in their genotypes .
Interestingly, we found that the intensity of pericarp color in black rice varies from dark purple to medium purple . Medium purple seeds of F1 and F2 plants showed purple color deposition in brown background. The purple pericarp progeny followed a segregation ratio of 1 dark purple (PpPp): 2 medium purple (Pppp): 1 brown (pppp), suggesting that the dominant Pp allele was incomplete over the recessive pp allele of the Pp gene (Fig, 5). Based on the genotype analysis shown in figure 5, the pericarp color phenotypes of the F3 progeny match with the genotypes exactly (Fig. 6). A strong peak of cyanidin-3-O-glucoside (kuromanin) was observed in the dark purple pericarp rice having the Pb_PpPp genotypes. But, cyanidin-3-O-glucoside peak was detected significantly lower in the medium purple pericarp rice (Pb_Pppp), and no cyanidin-3-O-glucoside peak was identified in the pericarp extraction of the brown pericarp rice (Pb_pppp) (Fig. 2). Hence, it is clear that the level of cyanidin-3-O-glucoside was determined by the number of copies of Pp alleles of the genotype in each progeny . Furthermore, the dominant Pp allele was found incompletely dominant over the recessive pp allele in rice and the concentration of cyanidin-3-O-glucoside in the pericarp of black rice also affected by the number of dominant Pp alleles .
Fig. 6.Analysis of the allelic polymorphism among progeny of the crosses between a black pericarp rice and a white pericarp rice. (A) DNA sequence fragments of the amplified Pb gene (OsB1 DNA sequence). The black pericarp rice ‘Hugnambyeo’ was composed of two homologous dominant Pb alleles and presented a BamH1 restriction enzyme site in the amplified DNA sequences of each corresponding allele. The white pericarp rice ‘Hwayoungbyeo’ was composed of two homologous recessive pb alleles without any BamH1 restriction enzyme site in the amplified DNA sequences. The pb allele of the white pericarp rice has a 2bp (GT) insertion in the BamH1 site of the same DNA sequences in the Pb allele. Schematic drawings are presented after BamH1 digestion in the DNA of the Pb allele resulting in division into two fragments of DNA. (B) Genetic analysis of the genotypic and phenotypic constitutions of the F3 progeny of the crosses between a black pericarp rice and a white pericarp rice. Phenotypes are indicated by the pericarp colors, with DP indicating dark purple, MP indicating medium and mixed purple, Wh indicating white, and Br indicating brown. Genetic analysis of Prp trait was approved that phenotypes of progenies has been corresponded completely to their genotypes .
Accumulation of anthocyanins in rice pericarp during grain development
The green pigments accumulated in seed from fertilization to dough stage followed by continuous degradation of the deposited green pigments which finally disappeared in mature seed of white rice. Fading or disappearing of green pigment in pericarp of white rice by 25-30 days after pollination (DAP) in field conditions were also reported . When green pigment in seed started to degrade, color pigments started to accumulate simultaneously at the beginning of the desiccation stage and continued to intensify up to physiological maturity of seed development . Brown pigment deposition was started at 15 DAP adjacent to the embryo and the entire seed turned into brown color followed by the complete disappearing of green pigment at 25 DAP in the brown pericarp rice. Furthermore, brown pigment accumulation continued to intensify the color of deep brown pericarp rice seed at harvest. In purple pericarp rice, the pigments accumulation was occurred in the pericarp at the beginning of the dough stage and persistently increased the accumulation of purple pigments until physiological maturity and begun to decrease slightly until at harvest.
An increasing trend of seed chlorophyll content was observed from 5th day after pollination (DAP) which reached to maximum level at 10th DAP and thereafter decreased sharply. The highest amount of chlorophyll were recorded in white rice which were found statistically identical with brown rice, whereas the lowest chlorophyll content was observed in black rice both at 10 DAP and 15 DAP (Fig. 7A). Grain anthocyanin content of purple pericarp rice was revealed much higher than the brown and white pericarp rice throughout the growth season of rice (Fig. 7B). A gradual increase in anthocyanin content was observed from 5th DAP and the whole rice seed become purple in color after 15 DAP. Furthermore, the anthocyanin content of purple pericarp rice was reached maximum at 25 DAP followed by a gradual decrease until harvest. Finally, the endosperms of black, brown and white rice were appeared fade purple, fade brown and white color, respectively.
Fig. 7.Changes in chlorophyll and anthocyanin contents of seed extracts from the purple, brown and white pericarp rice. (A) Changes in grain chlorophyll content. (B) Changes in grain anthocyanin (C) Changes in chlorophyll content of flag leaves. (D) Changes in grain filling rate (mg/p.d.) .
Anthocyanins pigment accumulation in pericarp decrease rice yield
The yield of colored rice is much lower compared to hybrid and white rice . Significantly lower seed weight of rice having purple pericarp in compare with rice having white pericarp was also reported . We also hypothesized that the lower seed weight of purple pericarp rice might be due to the accumulation of anthocyanin in pericarp . As we observed that pigments deposition in colored rice may be the reason for decrease of chlorophyll content in spikelet (Fig. 7A), which could be responsible for reduced yield . In our previous study, inverse relationship between chlorophyll and anthocyanin content in the pericarp were found . The probable reasons may be the deposition of carbohydrates stimulates anthocyanin metabolism and chlorophyll degradation . There is a significant relationship between seed length and seed weight. The reduced sink size may be resulted in the decreased 100 seed weight, which suggested that purple pericarp rice possessed reduced sink size (Fig. 8). It was also reported that purple pericarp rice found to give low yield as compared with white pericarp rice that might be due to reduced sink size . The reduced grain thickness and lower yield in black rice near isogenic lines (NILs) found in Japan . So, the small seed size of black rice resulting in its reduced yield potential may not be correlated with yield related genetic traits rather than it may related to physiological factors associated with anthocyanin synthesis in pericarps. These factors would reduce the photosynthetic rate and grain filling rate in seed which ultimately reduced the yield of black rice .
Fig. 8.Comparison of seed length (mm), seed width (mm) and 100 seed weight (g) of the purple, brown and white pericarp rice .
Conclusion and future prospects
Anthocyanins are a major group of flavonoid compounds which are fundamentally responsible for the purple color of rice tissues and organs. There is an increasing trend to develop functional food rich in anthocyanin. To fulfill the increasing demand, the identification of regulatory proteins should be conducted together with the investigation of the parameters controlling their expression, and a better understanding of the regulatory mechanism involve in tissue specific anthocyanin synthesis in rice are the key prerequisite. As well, the endogenous factors which trigger the expression of the regulatory genes should also be determined. Mark genes of allelic variabilities are useful for black rice quality improvement. Understanding about the accumulation of anthocyanin during chlorophyll degradation, distribution of assimilates, source and sink relationship as well as grain development and yield of black rice is essential.