Phenotypic Characterization of the pg Mutant
The pg mutant plants showed purple gradient grain hulls, whereas the wild-type (WT) hulls were straw-white at the heading stage (Fig. 1a). The hulls of the pg mutant were straw-white at the initial heading stage (pg-0d), gradually turned pink at 10 days after heading (pg-10d), deepened to dark purple at 20 days after heading (pg-20d), and finally turned to brownish-yellow at the fully mature stage of the rice grains (30 days) (pg-30d) (Fig. 1b). Differences between WT and pg mutant were observed in the plant, spikelet, and grain traits, with higher panicle number per plant, higher seed setting rate, lower 1000-grain weight, and lower total grain number per panicle recorded in the pg mutant compared with those of WT plants (Table 1). There were no significant differences between the WT and pg mutant for single panicle weight, filled grain number per panicle, grain density, average panicle length, and plant height (Table 1).
Rice grain hull color is an easily observable trait and is a crucial morphological marker for rice breeding. The main rice hull color mutants are golden yellow (Wang et al. 2017), brown (Shao et al. 2012; Xu et al. 2015), and virescent (Wang et al. 2021), while the black mutant is one of the common wild rice traits (Zhu et al. 2011). The pg mutant is a novel rice hull color with ornamental value for the integration and development of agriculture and tourism. The accumulation of anthocyanins and the lack of lignin synthesis both contributed to the change of rice hull color, but the lack of lignin synthesis also caused the variation of internode color (Wang et al. 2017; Zhang et al. 2006). Therefore, the variation in pg hull may be due to the accumulation of anthocyanin derivatives.
Flavonoids Metabolic Profiling of the pg Mutant
Flavonoids comprise the majority of pigment molecules in rice hulls. A new metabolomic strategy based on UPLC-MS/MS was used to identify and estimate flavonoid metabolism (Chen et al. 2013; Peng et al. 2017), to assess the changes in flavonoid metabolites of pg mutant hulls at different developmental stages. Results revealed 217 flavonoids, including 46 flavonols, 73 flavones, 5 isoflavones, 18 anthocyanins, 40 flavone C-glycosides, 21 dihydroflavonols, 11 flavanols, and 3 chalcones in hulls from four heading stages (Additional file 3: Table S2). Among the 18 anthocyanins, 16 were identified in the straw-white hulls, 18 in the pink and purple hulls, and 17 in the brownish-yellow hulls (Additional file 3: Table S2), suggesting that colorless rice hulls can also synthesize anthocyanins.
Hierarchical cluster analysis was performed on the above profiles to evaluate differences between metabolic profiles across four developmental stages. The metabolite profile was divided into four major clusters: clusters I, II, III, and IV, representing the accumulation of flavonoids at pg-0d, pg-10d, pg-20d, and pg-30d, respectively (Fig. 2a). In addition, principal component analysis (PCA) was conducted to resolve the intrinsic structure of flavonoids variation in the relative content of flavonoids in hulls from four developmental stages. Clear metabolite separation of pg-0d, pg-10d, pg-20d, and pg-30d was observed through PCA, indicating significant intergroup specificity of flavonoids metabolites in the hulls of pg mutant at different developmental stages (Fig. 2b).
Orthogonal projection to latent structure discriminant analysis (OPLS-DA), a supervised pattern recognition method, enabled visualization and depiction of general variations in metabolism among the four groups. High predictability (Q2) and strong goodness of fit (R2X, R2Y) of OPLS-DA models were observed in the comparison between pg-0d and pg-10d (R2X = 0.974, Q2 = 1, R2Y = 1), pg-20d and pg-10d (R2X = 0.937, Q2 = 1, R2Y = 1), and pg-30d and pg-20d (R2X = 0.981, Q2 = 1, R2Y = 1), suggesting that the model is stable, reliable, and has good discriminant analysis ability (Fig. 2c–e). After 200 permutation test results of the OPLS-DA model, the R2’ and Q2’ of the new model were smaller than those of the original after Y replacement (Additional file 1: Fig. S1), indicating that the differential metabolites between different groups could be screened according to their variable importance in the project (VIP).
Identification of Differential Flavonoids Metabolite
To understand the metabolic differences between pg-0d, pg-10d, pg-20d, and pg-30d, a differential metabolite screen was run among 217 identified metabolites based on fold change (FC) and VIP (FC ≥ 2 or ≤ 0.5 and VIP ≥ 1.0 were set as thresholds). Based on this criterion, there were 53 differential metabolites (50 upregulated and 3 downregulated) between pg-0d and pg-10d, 47 (25 upregulated and 22 downregulated) between pg-10d and pg-20d, 43 (all downregulated) between pg-20d and pg-30d, and 48 (19 upregulated and 29 downregulated) between pg-0d and pg-30d (Additional file 1: Fig. S2).
The differential metabolites between the four developmental stages were mapped using the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). Anthocyanin synthesis was the most significantly enriched metabolic pathway in pg-10d and pg-20d groups, accounting for 31.25% and 29.41%, respectively (Fig. 3a, b). Furthermore, in the pg-10d and pg-20d groups, delphin chloride, peonidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-glucoside, and pelargonidin 3-O-glucoside metabolites were upregulated in the anthocyanin synthesis pathway compared to the pg-0d group. However, in the pg-30d group, only one metabolite was associated with anthocyanin synthesis (Fig. 3c), indicating that anthocyanin metabolism was the main cause of the color change in the pink and purple hulls.
Comprehensive Comparison of the Metabolism of Flavonoids in Grain Hulls
Anthocyanins are a branch of flavonoid biosynthesis. Colored flavonoids (flavanols, isoflavonoids, and flavones) and their glycosides are responsible for coloring leaves, fruits, and flowers (Zhang et al. 2020; Berni et al. 2021; Yang et al. 2022). Analysis of the relative contents of the top 20 metabolites in rice hulls at different developmental stages revealed that tricin 4′-O-β-guaiacylglycerol had the highest relative content in pg-0d (straw-white) (flavones, 53.4 × 106) and pg-30d (brownish-yellow) (flavones, 28.5 × 106), followed by salcolin A [flavones, 47.7 × 106 (pg-0d, straw-white) and 27.6 × 106 (pg-30d, brownish-yellow)] (Table 2). However, cyanidin O-syringic acid was the most abundant substance in pg-10d (pink) (anthocyanins, 66.2 × 106) and pg-20d (purple) (anthocyanins, 68.0 × 106), followed by tricin 4′-O-β-guaiacylglycerol [flavones, 60.7 × 106 (pg-10d, pink) and 49.4 × 106 (pg-20d, purple)] (Table 2). Previous studies found that cyanidin O-syringic acid was the most abundant anthocyanin in red- and purple-colored vegetables and fruits, such as kiwifruit, kiwiberry, and radishes (Montefiori et al. 2009; Yu et al. 2020; Zhang et al. 2020). However, contrary to previous studies, cyanidin 3-O-glucoside and peonidin 3-O-glucoside were the two major anthocyanins found in the pericarp of black rice (Lee 2010; Shao et al. 2013; Das et al. 2020).
Chalcone synthase catalyzes the combination of p-coumaroyl-CoA with three acetate units from malonyl-CoA to produce tetrahydroxychalcone (anthocyanin skeleton) (Saigo et al. 2020; Xia et al. 2021). However, the conversion of the yellow-colored tetrahydroxychalcone into colorless naringenin is catalyzed by the chalcone isomerase enzyme. In addition, naringenin subsequently produces apigenin, hesperetin, genistein, and dihydrokaempferol via FNS, MTs, and F3H respectively (Cappellini et al. 2021). Finally, the three colorless substances are converted to flavones, flavonols, and anthocyanins (Cappellini et al. 2021). As shown in Fig. 4, tetrahydroxychalcone and naringenin contents were similar in straw-white and pink hulls but were upregulated in purple hulls. The relative contents of hesperetin O-malonylhexoside, apigenin-7-O-(6′-O-acetyl)-β-D-glucoside, apigenin 7-rutinoside, apigenin-6-C-glucose-8-xylcose, apigenin-8-C-glucoside, apigenin 8-C-pentoside, genistein 7-glucoside, genistein 8-C-apiosylglucoside, genistein 8-C-glucoside, and kaempferol 3-O derivatives were consistently downregulated during hull development (Fig. 4). In contrast, kaempferol 7-O-glucoside was consistently upregulated during hull development. Furthermore, 12 anthocyanins (6 cyanidin, 4 peonidin, 1 pelargonidin, and 1 delphin) were upregulated in pink, purple, and brownish-yellow hulls (Fig. 4; Additional file 4: Table S3). Cyanidin 3-O-malonylhexoside and delphin chloride were upregulated more than 1000-fold. However, cyanidin 3-O-malonylhexoside was abundant only in pink and purple hulls, indicating that the change in cyanidin 3-O-malonylhexose led to the pg phenotype of rice hulls (Fig. 4; Additional file 4: Table S3). The results indicated that the synthesis pathways of flavones and flavonols were mimicked during the development of pg mutant hulls; in contrast, the synthesis pathways related to cyanidin were promoted.
Genetic and BSA Correlation Analysis
To clarify the pg mutant regulatory genes, BSA-seq was used to perform gene mapping. All F1 plants derived from the crossing of the pg mutant and the Ziyedao (green grain hull) (Oryza sativa L. subsp. japonica) uniformly displayed pg mutant hulls. Among 557 F2 plants, 426 were purple gradient, and 131 showed green hulls. As segregation in the F2 population displayed a good fit of 3:1 ratio (χ2(3:1) = 0.652 < χ2(0.05) = 3.84), the pg grain hull trait in pg mutant was controlled by one nuclear dominant gene.
Furthermore, 2,072,328 SNPs were obtained by simplified genome sequencing of the pg mutant and green hull DNA pools. After eliminating the less reliable markers, 898,837 high-quality SNPs with uniform coverage of 12 rice chromosomes were obtained. The ΔSNP index was then fitted using the DISTANCE method, and the association threshold was obtained by combining the theoretical segregation ratio of the population to 0.667. As a result, one interval was associated with chromosome 4, 14.22 Mb long, containing 2209 genes, of which 789 had non-synonymous mutation loci (Fig. 5a). Furthermore, the ED values were analyzed by counting the depth of each base in the different mixing pools and calculating the ED values for each site. Finally, the median + 3SD = 0.60 of the fitted values for all loci was taken as the association threshold for the analysis. Based on the association threshold, one interval 11.57 Mb in length was obtained on chromosome 4, containing 1,847 genes, of which 747 had non-synonymous mutation loci (Fig. 5a).
Gene Mapping of the pg Mutant
The screening of molecular markers within the BSA association interval for genotypic validation of both parents and the F2 population showed that the gene for pg hulls was detected on chromosome 4 between 4–83.5 M and 4–99.3 M. For further mapping, the plants with purple gradient hulls were used to trap the target gene by narrowing the distance between 4–83.5 M and 4–99.3 M. Finally, the target gene was narrowed down to a interval between markers RM17321 and 4–94.4 M. The genetic distance between the two markers was about 2.0 cM, and the physical distance was approximately 1.38 Mb (Fig. 5b). The mapped region contained 154 putative genes, of which 4 genes, including Os04g0557200 encoding an anthocyanin regulatory R-S protein, Os04g0557500 encoding a bHLH transcription factor, Os04g0557800 similar to a R-type bHLH protein, and Os04g0565900 containing a bHLH domain were predicted to be associated with flavonoid synthesis.
The C–S–A gene system regulates rice hull color, involving C1 encoding the R2R3 MYB transcription factor, S1 encoding the bHLH protein and functioning tissue-specific, and A1 encoding a dihydroflavonol reductase has been proposed (Sun et al. 2018; Qiao et al. 2021). A protein–protein interaction occurs between the bHLH and R2R3 MYB domains, activating downstream genes in the structural anthocyanin biosynthesis pathway (Kim et al. 2021; Kong et al. 2012). Alterations to the HLH domain can affect protein–protein interactions between HLH and any other protein, enhancing or reducing the activities of bHLH proteins (Kim et al. 2021). In this study, BSA-seq and gene mapping approaches were used to map the candidate gene to a 1.38 Mb region on chromosome 4. In the mapped region, four genes, Os04g0557200, Os04g0557500, Os04g0557800, and Os04g0565900, were associated with flavonoid synthesis. Os04g0557200, encoding an anthocyanin regulatory R-S protein, was expressed specifically in fills, buds, and mammary grains (Wang et al. 2015). Os04g0557500 is presumed to be a candidate gene for hull-specific pigmentation (Sun et al. 2018). C1 interacts with S1 and activates A1 expression resulting in cyanogenic 3-O-glucoside accumulation (Sun et al. 2018). However, in our study, cyanidin O-syringic acid showed the highest pigmentation in pg grain hulls. Therefore, further studies are needed to validate the regulatory genes of the pg grain hulls.
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