Introduction

Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants. Several different types of plant photoreceptors receive light signals and activate various signal transduction pathways to regulate flavonoid biosynthesis under the light condition (Peng et al. 2013; Jaakola 2013). CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiquitin ligase, acts as a central regulator in light signal transduction and light-induced anthocyanin biosynthesis pathways downstream of multiple light receptors by promoting the ubiquitination and degradation of various transcription factors (TFs), such as myeloblastosis (MYB), ELONGATED HYPOCOTYL5 (HY5), and HY5 HOMOLOG (HYH); repressing the expression of structural genes involved in the anthocyanin biosynthesis pathway; and thus suppressing anthocyanin biosynthesis (Maier et al. 2013; Ang et al. 1998; Holm et al. 2002). The mechanisms and regulation patterns associated with COP1-mediated or light-induced anthocyanin biosynthesis have been extensively studied in many plant species, including the model plant Arabidopsis thaliana (Maier et al. 2013), eggplant (Solanum melongena L.) (Jiang et al. 2016), apple (Malus domestica) (Li et al. 2012), pear (Pyrus bretschneideri) (Wu et al. 2019), litchi (Litchi chinensis Sonn.) (Zhang et al. 2016), and sweet cherry (Prunus avium L.) (Liang et al. 2020). However, the COP1-mediated light signaling pathway that regulates flavonoid accumulation in rice (Oryza sativa L.) grain has not yet been fully elucidated.

Glycosylation is one of the most common modifications of flavonoids (Alseekh et al. 2020), and some cereal crops, including rice and wheat (Triticum aestivum L.), predominantly accumulate flavonoids in the C-glycosylated form (Brazier-Hicks et al. 2009). The biosynthetic pathways of various flavonoids, including C-glycosyl flavonoids, have been defined in many plant species. In higher plants, flavonoids are usually synthesized via the general phenylpropanoid pathway, in which phenylalanine is converted into 4-coumaroyl-CoA by three enzymes including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate: coenzyme A (CoA) ligase (4CL). The first committed step in the flavonoid biosynthetic pathway is catalyzed by chalcone synthase (CHS), which produces naringenin chalcone. Subsequently, chalcone isomerase (CHI) catalyzes the isomerization of chalcone to form naringenin, the common precursor of various classes of flavonoids (Winkel-Shirley 2001). In the following reaction, flavanone-3′-hydroxylase (F3’H) generates eriodictyol using naringenin as a substrate, and then flavanone 2‐hydroxylase (F2H) converts naringenin or eriodictyol to the corresponding 2-hydroxyflavanones. In the C-glycosyl flavone biosynthesis pathway, C-glycosyl-2-hydroxyflavanones are generated by C-glycosyltransferase (CGT) which catalyze the UDP-glucose-dependent C-glycosylation. In the last step, C-glycosyl flavones are produced by dehydratase, which selectively converts C-glucosyl-2-hydroxyflavanones to C-glycosyl flavones (Du et al. 2010; Brazier-Hicks et al. 2009).

It is well-known that the flavonoid biosynthetic pathway is mainly regulated by TFs, including a R2R3-MYB, a basic helix-loop-helix (bHLH), and a WD40-repeat protein (Ramsay and Glover 2005). Together, these proteins form the MYB-bHLH-WD40 (MBW) complex, which binds to the promoters of structural genes that play a crucial role in the biosynthesis of flavonoids including anthocyanin and proanthocyanidin (Gonzalez et al. 2008; Xu et al. 2015; Li 2014). In rice, several regulatory genes encoding the components of the MBW complex have been identified. The R2R3-type MYB TFs, OsC1 (maize C1 homolog) is involved in anthocyanin accumulation in the leaf sheath and apiculus (Chin et al. 2016), and another MYB TF, OsKala3, is associated with anthocyanin biosynthesis in black rice pericarp (Kim et al. 2021b; Maeda et al. 2014). Among the bHLH-type TFs, Rc regulates proanthocyanidin biosynthesis in the pericarp of red rice grain (Sweeney et al. 2006), and OsB1 and OsB2/OsKala4 are responsible for anthocyanin biosynthesis in the purple leaf and black rice pericarp, respectively (Sakamoto et al. 2001; Oikawa et al. 2015). Oryza sativa TRANSPARENT TESTA GLABRA1 (OsTTG1), a WD40-repeat TF, is a crucial determinant of anthocyanin biosynthesis in various organs, such as hull, root, culm, grain, and leaf (Yang et al. 2021). Although these individual regulators have been identified, the comprehensive system regulated by MBW complexes for flavonoid biosynthesis in rice remains to be established.

The genetic regulation and molecular mechanism of embryo development have been intensively investigated in Arabidopsis, a dicot plant (ten Hove et al. 2015; Lau et al. 2012). Although many essential processes responsible for embryo development, including the asymmetric division of cells, establishment of apical-basal and radical patterns, and differentiation of epidermal and vascular tissues, are conserved between monocots and dicots, a number of embryo developmental characteristics are distinct between monocots and dicots (Zhao et al. 2017). Unlike dicot embryo, the monocot embryo exhibits morphologically distinct embryonic organ types (such as scutellum, coleoptile, and coleorhiza) and organ number (such as cotyledons). Furthermore, the development of monocot embryo does not show a stereotypic cell division pattern and is not typically organized compared with that of dicot embryo. Thus, the morphology of suspensor is unclear, and apical and basal cell lineages are difficult to distinguish in monocot embryos. In addition to apical-basal polarity, the monocot embryo shows dorsal–ventral polarity during early embryogenesis (ten Hove et al. 2015; Zhao et al. 2017; Radoeva et al. 2019). For these reasons, rice embryogenesis exhibits biologically novel features compared with Arabidopsis embryogenesis. Consistent with the differences in embryo development between monocots and dicots, obvious differences in embryonic development and lethality have been observed between rice and Arabidopsis cop1 null mutants. While the Arabidopsis cop1 null mutant shows seedling lethality, the rice cop1 null mutant exhibits failed seed germination and unstructured embryo development (Kim et al. 2021a; Castle and Meinke 1994). Thus, the elucidation of genes that affect transcriptomic changes in the developing rice embryo will provide novel insights into embryogenesis in monocots or grasses, and will expand our understanding of the molecular mechanisms of COP1-related embryogenesis in rice.

Genes and their interactions responsible for photomorphogenesis and anthocyanin accumulation have been well-studied in the Arabidopsis cop1 mutant. However, we recently found that the newly isolated rice cop1 mutant exhibits distinct phenotypic characteristics, such as high-level C-glycosyl flavone accumulation and embryo lethality, compared with the previously reported Arabidopsis cop1 mutants (Kim et al. 2021a). Additionally, comprehensive transcriptomic changes caused by the mutation of OsCOP1, which is associated with seed pigmentation and embryogenic processes, remain still unknown. Here, we performed comparative transcriptome analysis of the oscop1 null mutant to investigate the gene families involved in C-glycosyl flavone biosynthesis and abnormal embryo formation during early seed development. This study provides transcriptomic evidence of how OsCOP1 mediates gene expression to regulate flavonoid biosynthesis and embryogenesis during seed development in rice.

Materials and Methods

Plant Materials and Sample Preparation

The yel-hc mutant was derived from japonica rice cultivar Hwacheong by N-methyl-N-nitrosourea (MNU) mutagenesis. The yel-hc mutant was fixed and maintained as a heterozygote, since the mutant allele is embryonic lethal in the homozygous state. Wild-type and yel-hc mutant plants were grown in the paddy field at the Experimental Farm of Seoul National University, Suwon, Korea. Harvested rice grains were air-dried, and the moisture content was reduced to approximately 13%. Samples were stored in an environmentally controlled room at 11 °C for 2 months, and then de-husked and hand-selected to eliminate cracked or discolored seeds. Each seed sample was ground using a mill (IKA A11B, Staufen, Germany) and sieved bypassing through a 300 μm filter prior to further experiments.

Measurement of Dehulled Grain, Embryo, and Endosperm Weights

Hundred-grain, -endosperm, and -embryo weights were measured (100 seeds × 3 replicates; 10% water content) using an analytical balance (CAS Corporation, NJ, USA). To measure the weight of embryos and endosperms, the embryos were dissected from grains and weighed separately. Phenotypic data collected from the yel-hc mutant and wild-type materials were statistically analyzed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

Embryo Microscopy

Dehulled yel-hc and wild-type mature seeds were incubated in water for 3 h at room temperature to soften the embryos. Then, the whole seed was longitudinally sliced in half, and the embryo was photographed using the HD’MEASURE software (HANA Vision Systems, Korea).

Total Flavonoid Content

Total flavonoid content was determined as described previously (Jia et al. 1999), with a slight modification. Briefly, 250 μL of crude extract was mixed with 1,250 μL of distilled H2O and 75 μL of NaNO2 (5%, w/v). After 5 min, 150 μL of 10% AlCl3·6H2O was added, and the mixture was left to settle for 6 min. Then, 500 μL of 1 M NaOH and 275 μL of distilled H2O were added to the solution, mixed well, and incubated at room temperature for 15 min. Then, the absorbance of the mixture was measured at 510 nm. A calibration curve was prepared using a standard solution of (+)-catechin hydrate, and the total flavonoid content was expressed as milligrams of catechin equivalents (CE) per 100 g of tissue dry weight (DW). Three independent biological extracts, each with three technical replicates, were analyzed.

RNA Isolation, Library Preparation, and Transcriptome Sequencing

Total RNA was extracted from 7-day-old seeds (7 days after pollination) of the wild-type and yel-hc mutant, with three biological replicates, using TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Japan), according to the manufacturer’s instructions. The cDNA libraries were prepared for 150 bp paired-end sequencing using TruSeq Stranded mRNA Sample Prep Kit (Illumina, CA, USA). Briefly, mRNA molecules were purified from 1 μg of total RNA using oligo (dT) magnetic beads. The purified mRNAs were fragmented, and first-strand cDNA was synthesized using random hexamer primers. Then, using the first-strand cDNA as a template, the second strand of cDNA was synthesized, generating double-stranded cDNAs. After the sequential steps of end repair, A-tailing, and adapter ligation, the cDNA libraries were amplified by PCR, and their quality was evaluated with Agilent 2100 BioAnalyzer (Agilent, CA, USA). The cDNA libraries were then quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA), according to the manufacturer’s protocol. Following cluster amplification of denatured templates, sequencing was performed using the Illumina NovaSeq 6000 platform (Illumina, CA, USA) in paired-end (2×150 bp) mode.

Transcriptome Analysis

Paired-end RNA-seq reads were initially aligned to the IRGSP-1.0 pseudomolecule/MSU7 (http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml) version of rice reference transcriptome sequences to determine inner distance between mate pairs (mate inner distance) using a homemade Python script. The RNA-seq reads were then realigned to the same version of rice reference genome using Tophat2 (v2.1.1) and Bowtie2 (v2.3.4.1), considering the reference gene annotation and distribution of mate inner distance to increase accuracy in transcriptome sequence alignment. The number of reads mapped to the coding sequence (CDS) of each gene was counted using HTSeq-count (v0.12.4) under the ‘union’ mode. Statistical significance of the differences in gene expression levels between the triplicate ‘wild-type’ and ‘yel-hc’ samples was examined using DESeq2 (v4.1), considering adjusted p-value (padj) less than 0.05. Genes differentially expressed between yel-hc and wild-type samples (log2 fold change [FC] > 2) were defined as differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis of the DEGs was implemented through the web-based Comprehensive Annotation of Rice Multi-Omics (CARMO) platform (http://bioinfo.sibs.ac.cn/carmo/) (Wang et al. 2015), with false discovery rate (FDR) < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) (Kanehisa and Goto 2000) pathway enrichment analysis of DEGs was performed using the TBtools software (Chen et al. 2020), with q-value < 0.05, to test the enrichment of DEGs in particular KEGG pathways. The categories of TFs in rice were obtained from the Plant Transcription Factor Database (PlnTFDB; http://plntfdb.bio.uni-potsdam.de/v3.0/ (Perez-Rodriguez et al. 2010) to analyze TF-encoding DEGs. The MapMan tool (http://MapMan.gabipd.org; version 3.5.1R2) was used to visualize the functional categories of DEGs in the pathways (Thimm et al. 2004). Heatmap visualization was carried out using the Clustvis tool (https://biit.cs.ut.ee/clustvis/) (Metsalu and Vilo 2015).

Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA samples were subjected to first-strand cDNA synthesis using M-MLV reverse transcriptase (Promega, Madison, WI, USA), and qRT-PCR was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, Japan) on a CFX96™ Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Primers used for qRT-PCR analysis are listed in Table S1. Expression levels of genes were normalized relative to that of ACTIN, a housekeeping gene. Data were analyzed using the comparative Ct method. Expression levels were compared using two-tailed Student’s t-test.

Results

Characterization of the Phenotypic Traits of yel-hc Seeds

The Hwacheong (japonica rice)-derived yellowish-pericarp embryo lethal (yel) mutant, yel-hc, displayed yellow and dark-brown to black coloration in the pericarp and embryo, respectively, because of high-level flavonoid accumulation (Fig. 1)a–d. Embryonic lethality was one of the most distinct characteristics of the yel-hc mutant compared with wild-type Hwacheong. To observe the intact mature embryo, the imbibed yel-hc and wild-type seeds were sliced longitudinally. Mature wild-type seeds showed normally developed embryos (Fig. 1)e, whereas yel-hc seeds contained abnormally differentiated embryos and indistinguishable embryonic organs including coleoptile, epiblast, radicle, and shoot apex, resulting in the appearance of irregular and unstructured organs in the embryo (Fig. 1f–i). In addition, dark green pigmentation was observed in yel-hc embryos, and the hundred-grain weight was significantly lower in yel-hc than in the wild-type (Fig. 1)j. Consistently, the hundred-endosperm and -embryo weights were also significantly lower in the yel-hc mutant than in the wild-type (Fig. 1)k and l. However, the relative endosperm weight, defined as the weight of the endosperm relative to that of the grain, was higher in yel-hc (97.8%) than in the wild-type (96.5%), whereas the relative embryo weight, defined as the weight of the embryo relative to that of the grain, was lower in yel-hc (2.2%) than in the wild-type (3.5%) (Fig. 1) k and l. These results indicate that the yel-hc mutation influences the overall development of seed; color of embryo and pericarp and weight of grain, endosperm, and embryo in rice.

Fig. 1
figure 1

Characterization of the phenotypic traits and total flavonoid contents of Hwacheong (wild-type) and yel-hc seeds. a, b Seeds of the wild-type (a) and yel-hc (b). c, d Embryos of the wild-type (c) and yel-hc (d). Scale bars = 1 mm. e–i Longitudinal sections of imbibed wild-type (e) and yel-hc (f–i) seeds. j–l Evaluation of 100 grain weight (j), 100 endosperm weight (k), and 100 embryo weight (l). m Total flavonoid contents of wild-type and yel-hc seeds. Cp coleoptile; Ep epiblast; Rd radicle; Sc scutellum; Sa shoot apex. Error bars represent standard deviation (SD) of three technical repeats. Asterisks indicate statistical significance, as determined by Student’s t-test (**p < 0.01; ***p < 0.001)

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Total Flavonoid Content of yel-hc Grain

Total flavonoid contents of yel-hc and wild-type grain extracts were assessed. The yel-hc grains showed a remarkably high total flavonoid content compared with wild-type grains (Fig. 1)m. The yel-hc grain contained 157.2 mg CE/100 g DW, which was approximately 60-fold higher than the total flavonoid content of wild-type grain, a significant difference (p < 0.001) (Fig. 1)m.

RNA-Seq and DEG Identification

To investigate the impact of mutation in the OsCOP1 gene on the transcriptome dynamics of rice seed, 7-day-old developing seeds of the wild-type and yel-hc mutant were subjected to RNA-seq. An average of 81.5 million paired-end reads, representing 96.6% of the total raw reads, were obtained from each sample, among which an average of 81.9% properly paired reads were mapped to the Nipponbare reference genome. A total of 27,810–28,493 expressed genes were detected in wild-type and yel-hc samples (Table S2). DEGs were selected based on two thresholds: | log2FC |> 2 and padj < 0.05. A total of 979 DEGs, including 577 upregulated and 402 downregulated genes, were identified (Fig. 2a and b).

Fig. 2
figure 2

Functional classification of differentially expressed genes (DEGs). a Volcano plot of genes differently expressed between wild-type and yel-hc seeds. The DEGs were identified based on two thresholds: |log2FC|> 2 and padj < 0.05. Red and blue dots represent significantly upregulated and significantly downregulated genes, respectively, and gray dots represent non-significant DEGs. b Numbers of upregulated and downregulated DEGs. c The 20 most enriched GO terms of DEGs (FDR < 0.05). d KEGG pathway analysis of DEGs. The size of each point represents the number of DEGs in the pathway, and the color of the point represents the -log10FDR or -log10q-value. Rich factor indicates the ratio of the number of DEGs to the number of genes annotated in the GO term or KEGG pathway

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GO Term and KEGG Pathway Enrichment Analyses of DEGs

GO term enrichment analysis was performed to classify the DEGs into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The results showed that DEGs were enriched in 15 GO terms in the BP category, four CC terms, and 16 MF terms (FDR < 0.05; Table S3). The top 20 enriched GO terms are shown in Fig. 2c. In the BP category, the most representative GO terms were ‘response to abiotic stimulus,’ ‘metabolic process,’ ‘oxidation–reduction process,’ and ‘biosynthetic process.’ In the CC category, DEGs were highly enriched in ‘cytoplasmic membrane-bounded vesicle,’ ‘cell,’ and ‘nucleus’ subcategories. In the MF category, the most significantly enriched subcategories were ‘sequence-specific DNA binding transcription factor activity,’ ‘oxidoreductase activity,’ ‘DNA binding,’ and ‘catalytic activity’ (Fig. 2c and Table S3). To further identify the functional pathways potentially involved in seed development in the rice cop1 null mutant, pathway enrichment analysis was performed based on the KEGG database. The results showed that 914 DEGs were assigned to 125 KEGG pathways (Table S4). Among them, DEGs were significantly enriched in seven pathways including ‘biosynthesis of other secondary metabolites,’ ‘transcription factors,’ ‘metabolism,’ ‘flavonoid biosynthesis,’ ‘cytochrome P450,’ ‘phenylpropanoid biosynthesis,’ and ‘prodigiosin biosynthesis’ (Fig. 2 d and Table S4). Most of the DEGs involved in KEGG pathways such as ‘biosynthesis of other secondary metabolites,’ ‘transcription factors,’ and ‘metabolism’ were assigned to the GO terms ‘metabolic process,’ ‘biosynthetic process,’ and ‘sequence-specific DNA binding transcription factor activity.’ Thus, KEGG pathway and GO term enrichment analyses provided further insight into the potential function of DEGs, which were predominantly associated with ‘biosynthesis of other secondary metabolites’ and ‘transcription factors’.

Metabolic Pathway Analysis of DEGs Using MapMan

To gain further insight into the metabolic and molecular factors affected by the mutation of OsCOP1, DEGs were analyzed using the MapMan tool. In the metabolism overview, a total of 76 DEGs were mainly assigned to cell walls, lipids, and secondary metabolisms. Most of the DEGs in the secondary metabolism pathway, which related to flavonoid and phenolic compound biosynthesis, were upregulated (Fig. 3a and Table S5). The predominance of secondary metabolism, cell wall, and lipid metabolism terms in MapMan analysis was consistent with the results of GO term and KEGG pathway enrichment analyses (Fig. 2c and d). In the regulation overview, the enriched DEGs were associated with TF, protein degradation, IAA and ethylene regulation, and receptor kinases. Notably, many genes related to ethylene signal transduction were upregulated. In particular, genes encoding MYB, bZIP, bHLH, AP2/EREBP, and homeobox TFs were the most abundant, indicating that these genes are largely involved in regulating flavonoid biosynthesis and embryogenesis in rice seeds (Fig. 3b and Table S6). The expression of genes belonging to large enzyme families, such as cytochrome P450, oxidases, UDP-glycosyltransferases, glutathione-S-transferases, GDSL-lipases, and peroxidases, showed predominant changes in yel-hc seeds. Many of the genes encoding cytochrome P450 proteins, oxidases, and UDP-glycosyltransferases were upregulated, whereas most of the DEGs encoding peroxidases were downregulated (Fig. S1a and Table S7). In addition, most of the DEGs involved in the biosynthesis of secondary metabolites, such as phenylpropanoids, lignin, lignans, and flavonoids, were significantly upregulated (Fig. S1b and Table S8). Taken together, these results suggest that flavonoid biosynthesis and embryo development are complex regulatory processes, involving transcriptional regulation and multiple metabolic pathways, in rice.

Fig. 3
figure 3

MapMan overview of changes in the expression of differentially expressed genes (DEGs) in yel-hc developing seeds. a Metabolism overview. b Regulation overview. The DEGs were binned into MapMan functional categories. Data represent log2FC values. Red and blue colors represent upregulated and downregulated genes, respectively

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Identification of Differentially Expressed TF Genes

Numerous molecular genetic studies have revealed that TFs play crucial roles in plant growth and development. In this study, we investigated TF genes associated with flavonoid biosynthesis and embryo development. Diverse families of TF-encoding genes were found to be differentially expressed; among 979 DEGs, 120 TF genes (66 upregulated and 54 downregulated) belonging to 33 different families were identified (Fig. 4a). Among the 66 upregulated TF genes, AP2-EREBP genes were the most abundant, followed by MYB, Orphans, bHLH, MYB-related, and WRKY. On the other hand, the TF gene family members, such as HB, bHLH, C2H2, OFP, and ABI3VP1, were mainly downregulated in the developing yel-hc seeds (Fig. 4a and Table S9). Among the identified TF genes, those encoding components of the MBW complex, which plays a central role in regulating flavonoid biosynthesis, were identified. The result revealed that all 10 differentially expressed TF genes putatively belonging to the MYB gene family were upregulated. Among these, two MYB TF genes, OsC1 and OsP1 (homolog of maize pericarp color 1), which act as crucial regulatory genes in anthocyanin and flavonoid biosynthesis pathways, were significantly upregulated (Fig. 4b and Table S9). Furthermore, 11 putative bHLH TF genes were identified, of which seven were downregulated and three were upregulated. In addition, expression levels of two WD40 genes, including OsRUP2 (LOC_Os02g02380), were significantly increased, while that of a putative WD40 gene was downregulated in yel-hc seeds (Fig. 4b). These results are consistent with the MapMan pathway analysis of DEGs, suggesting that the mutation of OsCOP1 affects the transcript levels of TF genes playing important roles in flavonoid biosynthesis and plant development.

Fig. 4
figure 4

Analysis of transcription factor (TF) family genes and MYB-bHLH-WD40 (MBW) complex genes among the differentially expressed genes (DEGs). a Differentially expressed TF genes. Numbers indicate the number of genes. b Heat map of the expression patterns of MBW complex genes. The color scale represents the log-transformed fragments per kilobase transcript per million mapped reads (FPKM) values. Gene IDs and names are shown on the right

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Analysis of Flavonoid Biosynthesis-Related Genes in yel-hc Seeds

The expression of genes related to main flavonoid biosynthesis was analyzed to explore genes responsible for the distinctive coloration of yel-hc seeds. First, we screened genes that function in the phenylpropanoid pathway to convert phenylalanine into 4-coumaroyl-CoA, which is ultimately utilized for flavonoid biosynthesis. Certain genes, including those encoding PAL (LOC_Os04g43800, LOC_Os02g41670), C4H (LOC_Os01g60450, LOC_Os02g26810), and 4CL (LOC_Os02g46970), were significantly upregulated in the developing yel-hc seeds (Fig. 5). Additionally, genes encoding CHS (LOC_Os11g32650) and CHI (LOC_Os11g02440, LOC_Os12g02370, LOC_Os03g60509), which are necessary to produce naringenin, were predominantly expressed in yel-hc. Interestingly, the expression level of genes involved in the C-glycosyl flavone biosynthesis pathway was significantly upregulated in yel-hc. For example, genes encoding F3'H (LOC_Os10g17260), F2H (LOC_Os06g01250), and CGT (LOC_Os06g18010) showed high expression levels in yel-hc. In particular, the expression level of OsCGT which catalyzes C-glycosylation to produce C-glycosyl flavones such as isoorientin and vitexin in the last step of the pathway, was dramatically increased in yel-hc. Genes encoding key flavonol biosynthetic enzymes, including F3H (LOC_Os03g03034) and FLS (LOC_Os02g52840 and LOC_Os01g61610), were also activated. By contrast, several major genes involved in anthocyanin and proanthocyanidins biosynthesis downstream of the flavonoid biosynthetic pathway, such as DIHYDROFLAVONOL 4-REDUCTASE (DFR), ANTHOCYANIDIN SYNTHASE (ANS), UDP-GLUCOSE FLAVONOID 3-O-GLYCOSYLTRANSFERASE (UFGT), LEUCOANTHOCYANIDIN REDUCTASE (LAR), and ANTHOCYANIN REDUCTASE (ANR), were downregulated or showed no significant difference in expression between wild-type and yel-hc seeds (Fig. 5). These results suggest that the mutation of OsCOP1 highly upregulated expression of genes involved in the flavonoid and C-glycosyl flavone biosynthetic pathways, resulting in the high-level accumulation of C-glycosyl flavones including isoorientin in yel-hc seeds.

Fig. 5
figure 5

Transcriptome profiling of genes involved in the flavonoid biosynthetic pathway. Heat map shows the changes in transcript levels of a representative subset of genes. Orange and blue colors indicate upregulation and downregulation, respectively. The number in the rectangle represents the log2FC value. Red and blue circles indicate significant (FDR < 0.05) and non-significant differences, respectively. PAL phenylalanine ammonia lyase; C4H cinnamate 4-hydroxylase; 4CL 4-coumarate: CoA ligase; CHS chalcone synthase; CHI chalcone isomerase; F3'H flavonoid 3'-hydroxylase; F2H flavanone 2-hydroxylase; CGT C-glycosyltransferase; DH dehydratase; F3H flavonoid 3-hydroxylase; FLS flavonol synthase; DFR dihydroflavonol 4-reductase; ANS anthocyanidin synthase; UFGT UDP-glucose: flavonoid 3-O-glucosyltransferase; LAR leucoanthocyanidin reductase; ANR anthocyanidin reductase; FNSII flavone synthase II; OMT O-methyltransferase; C5'H chrysoeriol 5'-hydroxylase

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Analysis of Embryo Development-Related Genes in yel-hc Seeds

Next, we analyzed the expression levels of previously reported embryogenesis-related genes. Some of the selected genes essential for embryo formation and development showing significant changes in expression (FDR < 0.05) are shown in Fig. 6. Consistent with the characteristics of yel-hc embryo, such as developmental defects and lethality, the expression level of developmental regulatory genes, such as auxin-signaling genes (OsPIN2, OsPIN1d, OsYUC7, and OsYUC9) and homeobox genes (OSH1, OSH15, OsWOX6, and OsWOX9C), were significantly downregulated in yel-hc (Fig. 6 and Table S10). Interestingly, transcript levels of genes involved in somatic embryogenesis (OsVP1, OsLEC1, OsLEC2, OsBBM1, OsBBM2, and OsBBM3) were significantly downregulated in yel-hc. In particular, expression levels of OsBBM3 and OsLEC1 were dramatically reduced in yel-hc (Fig. 6 and Table S10). Among the reported embryogenesis-related TF genes, the expression of NAC DOMAIN-CONTAINING PROTEIN 7 (OsNAC7), GROWTH-REGULATING FACTOR 10 (OsGRF10), and BZIP PROTEIN 8 (OsBZ8) was significantly downregulated in developing yel-hc seeds. Among the 15 differentially expressed late embryogenesis abundant (LEA) genes, 12 including OsEM were downregulated, and three were upregulated. Furthermore, some embryo development-related genes, such as AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE 2 (OsACO2), LIPID TRANSFER PROTEIN 2.1 (OsLTP2.1), and ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 43 (OsC3H43), were downregulated, whereas GIBBERELLIN 20-OXIDASE 1 (OsGA20ox1) and ALPHA-AMYLASE 1A (OsAmy1A) were significantly upregulated in yel-hc (Fig. 6 and Table S10). These results indicate that mutation in OsCOP1 influences the overall embryo formation and development processes in the developing seed.

Fig. 6
figure 6

Heat map of the expression patterns of embryogenesis-related genes. The color scale represents log-transformed fragments per kilobase transcript per million mapped reads (FPKM) values. Gene IDs and names are shown on the right

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Validation of RNA-Seq Data

The transcriptional profiles of 23 putative genes involved in flavonoid and anthocyanin biosynthesis, embryogenesis, and COP1-mediated signal transduction pathway were further validated by qRT-PCR. The dramatic upregulation of flavonoid/C-glycosyl flavone biosynthetic genes and downregulation of all zygotic/somatic embryogenesis-related genes, except OsAmy1A, observed in RNA-seq was confirmed by qRT-PCR. Similarly, the highly upregulated expression of OsCOP1-mediated light signaling transduction genes, OsHY5 and OsRUP2 observed in the RNA-seq was validated by qRT-PCR analysis (Fig. 7a). Thus, most of the genes, except OsANR, exhibited similar expression patterns as both RNA-seq and qRT-PCR analyses, with a strong correlation (Pearson correlation coefficient; R = 0.97) (Fig. 7b). However, among the four anthocyanin biosynthesis genes including OsDFR, OsANS, OsLAR, and OsANR, which showed no significant difference between WT and yel-hc in the RNA-seq analysis, OsANR was slightly but significantly downregulated in yel-hc in the qRT-PCR analysis (Fig. 7b). These results indicate that the transcriptomic profiling was accurately conducted and offered reliable explanation to correspond the response of mutation in OsCOP1 in transcriptome level.

Fig. 7
figure 7

Validation of the RNA-seq data of crucial genes by quantitative real-time PCR (qRT-PCR). a Comparison of log2FC values of genes between qRT-PCR and RNA-seq results. In RT-qPCR analysis, log2FC values were calculated using the 2−ΔΔCt method, and data were expressed as mean ± SD of three biological replicates. Asterisks indicate statistical significance, as determined by Student’s t-test (*p < 0.05; **p < 0.01). NS, not significant. b Correlation between qRT-PCR and RNA-seq data. The log2FC values of each gene obtained using the two methods were compared

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Discussion

COP1 is an E3 ubiquitin ligase and plays an important role in flavonoid biosynthesis by regulating light signal transduction in plants. A high level of anthocyanin accumulation has been reported in the cotyledons and leaves of Arabidopsis cop1 mutants including cop1-5 (Castle and Meinke 1994; Deng et al. 1991). In the cop1 mutant, the two R2R3-MYB TFs, PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2, are required for anthocyanin biosynthesis and the expression of structural genes. The activation of PAP1 and PAP2 is regulated by the COP1/SPA E3 ubiquitin ligase complex via HY5 in a light-dependent manner (Maier et al. 2013; Shin et al. 2013). Homologs of AtPAP1 (AtMYB75) gene have been identified in other plant species, such as tomato, sweet potato, grapevine, and apple, but no AtMYB75 homolog has yet been identified in rice, since AtMYB75 is a dicot-specific gene (Zhao and Bartley 2014). In the current study, ten MYB genes were identified as DEGs, and two of these genes, OsC1 (LOC_Os06g10350) and OsP1 (LOC_Os03g19120), which are well-defined as flavonoid regulatory genes, were highly upregulated in 7-day-old yel-hc seeds (Fig. 4 and Table S9). The P1 gene, which encodes an R2R3-MYB TF, is known to participate in the regulation of flavonoid biosynthesis in plants. Characterization of rice R2R3-MYB genes revealed that OsP1 (LOC_Os03g19120) is most closely related to subgroup 7 (SG7) MYBs including the maize gene ZmP1 and Arabidopsis genes AtMYB11, AtMYB12, and AtMYB111 (Zheng et al. 2019). In maize, ZmP1 primarily expresses in floral tissues, such as pericarps, cob glumes, and silks, and promotes the accumulation of phlobaphenes, C-glycosyl flavones, and maysin by activating the expression of a subset of flavonoid biosynthesis genes (Casas et al. 2016; Zhang et al. 2003; Grotewold et al. 1994; Falcone Ferreyra et al. 2013).

Another R2R3-MYB TF gene, C1, is known as a crucial regulator of anthocyanin biosynthesis in plants. In rice, several studies demonstrated that OsC1, a homolog of ZmC1, mainly regulates anthocyanin accumulation but not flavonoid accumulation in apiculus, stigma, hull, and leaf sheath by coregulating anthocyanin biosynthesis together with other anthocyanin biosynthesis genes such as OsDFR, OsPa, and OsRb (Saitoh et al. 2004; Sun et al. 2018; Chin et al. 2016; Meng et al. 2021; Zheng et al. 2019). However, it is likely that OsC1 is not associated with anthocyanin biosynthesis in rice pericarp (Zheng et al. 2019). According to Zheng et al. (2019), most cultivated rice lost their ability to synthesize anthocyanins because of negative selection during domestication. Cultivated rice accessions, which possess null mutations in certain anthocyanin biosynthesis genes, such as OsC1 and OsRb, were artificially selected by humans, although the reasons for selection against anthocyanin biosynthesis are unclear (Zheng et al. 2019). Hwacheong, the progenitor of yel-hc, is a typical japonica cultivar with white pericarp and non-pigmented leaves. It is likely that Hwacheong, like many other japonica cultivars, harbors non-functional alleles of the anthocyanin biosynthesis genes OsC1 and OsRb, which may explain the absence of anthocyanin biosynthesis in yel-hc seeds despite the high-level expression of OsC1. Therefore, these findings may suggest that OsP1 regulates the activation of an alternative flavonoid biosynthesis pathway in yel-hc seeds. Taken together, these results suggest that OsP1, rather than OsC1, may be a crucial regulator of a set of genes involved in the C-glycosyl flavone biosynthetic pathway in rice, and anthocyanin and C-glycosyl flavone biosynthetic pathways may be regulated by different regulatory genes or TFs in a tissue-specific manner in monocots and dicots.

In this study, we found that the biosynthesis of flavonoids, including C-glycosyl flavones, is regulated by MYB TFs and multiple regulatory factors. The expression of flavonoid biosynthetic genes, including OsCHS (LOC_Os11g32650), OsCHI (LOC_Os11g02440, LOC_Os12g02370, and LOC_Os03g60509), OsF3’H (LOC_Os10g17260), and OsF2H (LOC_Os06g01250), and a C-glycosylation pathway gene, OsCGT (LOC_Os06g18010), was significantly induced by the mutation of OsCOP1 in yel-hc. In particular, expression of OsCGT, which uses 2-hydroxyflavanones as substrates to generate C-glycosyl flavones, was dramatically upregulated (Fig. 5). By contrast, the expression level of structural genes involved in the biosynthesis of anthocyanins (OsDFR, OsANS, and OsUFGTs) and proanthocyanidins (OsLAR and OsANR) was downregulated in yel-hc, or there was no significant difference between WT and yel-hc seeds (Figs. 5 and 7). These results are consistent with our previous metabolite analysis, which showed that C-glycosyl flavones and their derivatives, including luteolin 6-C-glucoside (isoorientin), isoorientin 2"-O-glucoside, vitexin 2"-O-glucoside, isoscoparin 2"-O-glucoside, chrysoeriol 6, 8-di-C-hexoside, chrysoeriol 6-C-glucoside (isoscoparin), and apigenin 6-C-glucoside (isovitexin), accumulated to high levels in the embryo and endosperm of yel-hc. Among these, isoorientin, a CGT-catalyzed C-glycosyl flavone, was the most abundant flavone both in the embryo and endosperm of yel-hc; however, anthocyanins were not detectable in the embryo and pericarp of yel-hc (Kim et al. 2018). In addition, considerable research on flavonoid biosynthetic genes, controlled by regulatory genes, has been conducted in maize and rice, and the results of this research are consistent with those of this study. In maize, ZmP1 regulates the transcription of F3'H, which catalyzes the conversion of naringenin to eriodictyol by binding to F3'H promoter (Sharma et al. 2012), and directly targets F2H, which converts naringenin and eriodictyol into 2-hydroxynaringenin and 2-hydroxyeriodictyol, respectively, resulting in the accumulation of flavones (Morohashi et al. 2012). Similarly, OsP1 specifically activates upstream biosynthetic genes such as OsCHS, OsCHI, and OsF3'H, whereas OsC1 activates all anthocyanin biosynthetic genes including OsCHS, OsCHI, OsF3'H, OsF3H, OsDFR, and OsANS via the ternary MYB-bHLH-WD40 complex (OsC1–OsRb–OsPAC1) in rice leaves (Zheng et al. 2019). Therefore, our transcriptome profiling results demonstrate that the expression of flavonoid biosynthetic genes is upregulated by regulatory genes such as OsP1, and these biosynthetic and regulatory genes play an important role in flavonoid (C-glycosyl flavone) biosynthesis, but not anthocyanin biosynthesis, in the embryo and pericarp of rice cop1 null mutant. Further investigation is needed to elucidate a more accurate regulation of the different branches of flavonoid biosynthesis by MYBs or MBW complexes in the rice pericarp.

RNA-seq provides a comprehensive overview of several gene families involved in embryogenesis. Homeobox (HB) genes, which encode TFs that regulate the expression of genes involved in pattern formation, cell specification, and cell differentiation during embryogenesis, are thought to play roles in plant development. Sato et al. demonstrated that Oryza sativa homeobox1 (OSH1) and OSH15 encode KNOTTED1 (KN1)-like homeobox (KNOX) protein involved in the formation and maintenance of shoot apical meristem (SAM) (Sato et al. 1996, 1998), while the osh1 osh15 (d6) double mutant embryo failed to form SAM (Tsuda et al. 2011). Other KNOX genes, such as OSH6 and OSH71, were highly expressed in the central part of the embryo at the globular embryo stage during rice embryogenesis (Sentoku et al. 1999). In the present study, the expression level of OSH1 and OSH15 was strongly reduced in the 7-day-old developing seeds of yel-hc. In addition, the expression levels of OSH6 and OSH71 were also significantly downregulated in yel-hc, although not to the same extent as that of OSH1 and OSH15 (Fig. 6 and Table S10). These results imply that the downregulated expression of these HB genes is one of the main causes of the abnormal differentiation of embryo organs, especially in the SAM region of yel-hc.

WUSCHEL-related homeobox (WOX) proteins, which comprise a plant-specific subclade of the HB TF family, play critical roles in early embryogenesis. WOX2 and WOX8 induce distinct cell fates after the asymmetric zygotic division by restoring their expression to the apical (WOX2) and basal (WOX8) lineages. In addition, WOX8 and its closest homolog, WOX9, redundantly affect normal development in both the basal and apical embryo lineages and promote PIN-FORMED 1 (PIN1) expression in the proembryo of Arabidopsis (Breuninger et al. 2008). The maize homologs of AtWOX8 and AtWOX9, ZmWOX9A and ZmWOX9B, respectively, are expressed at higher levels in the basal part than in the apical part, indicating that these genes are potentially involved in axis formation during early embryo patterning, as in Arabidopsis (Chen et al. 2017). In rice, WOX genes are involved in root apical meristem (RAM) specification and maintenance (QHB/OsWOX5) (Kamiya et al. 2003), meristem differentiation (OsWOX4) (Ohmori et al. 2013), crown root development (OsWOX11) (Zhao et al. 2009), and tiller growth (DWT1/OsWOX7) (Wang et al. 2014). In the present study, the expression of OsWOX6, OsWOX9C, and OsWOX10 was significantly downregulated in yel-hc (Fig. 6 and Table S10). These three WOX genes support the results of a previous study, which showed that WOX genes are involved in developmental regulation during early embryogenesis in rice. Among them, OsWOX9C, which is closely related to AtWOX8 and AtWOX9, showed biased expression along the apical-basal axis, and OsWOX6 and OsWOX10, which are phylogenetically close to AtWOX11 and AtWOX12 (Lian et al. 2014), exhibited biased expression along the dorsal–-ventral axis in rice embryo at 3 days after pollination (DAP) (Itoh et al. 2016).

PIN genes, involved in auxin efflux, are necessary for establishing apical-basal polarity during early embryogenesis. PIN1, PIN3, PIN4, and PIN7 are expressed in the embryo in Arabidopsis (Friml et al. 2003; Adamowski and Friml 2015). Analysis of the spatial expression pattern of PIN-like genes revealed that OsPIN1, OsPIN2, OsPIN5, and OsPIN8 showed polarized expression along the apical-basal axis in 3-DAP rice embryo (Itoh et al. 2016). In our study, the expression of OsPIN1b, OsPIN1d, and OsPIN2 was significantly downregulated in yel-hc (Fig. 6 and Table S10). These findings serve as reliable evidence supporting the role of OsPINs in auxin-dependent polarity determination during early embryogenesis in rice. Altogether, these results indicate that COP1 has a wide influence on embryo development, and that the embryo defects of yel-hc originate during embryogenesis, when SAM differentiation, axis formation, and polarity determination are not established correctly. This is because of the insufficient expression of HBs, WOXs, and PINs, although the encoded proteins are functionally redundant.

Aside from zygotic embryogenesis-related genes, several somatic embryogenesis-related genes, such as BABY BOOM (BBM) (Boutilier et al. 2002), LEAFY COTYLEDON (LEC) (Lotan et al. 1998; Stone et al. 2008), FUSCA3 (FUS3) (Luerssen et al. 1998; Ledwon and Gaj 2011), ABA INSENSITIVE3 (ABI3) (Parcy et al. 1994), WUSCHEL (WUS) (Zuo et al. 2002; Lou et al. 2022), WOX (van der Graaff et al. 2009), EMBRYOMAKER (Tsuwamoto et al. 2010), SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) (Hecht et al. 2001), and AGAMOUS-LIKE 15 (AGL15) (Thakare et al. 2008), which regulate plant cell identity and development, somatic embryogenesis, and seed development, have been identified in plants. However, some of these genes, including BBM and LEC, exhibit overlapping functions during embryo formation, such as polar transport and auxin gradient, and are directly or indirectly linked to zygotic embryogenesis-related genes in the molecular network (Dodeman et al. 1997; Horstman et al. 2017). In this study, OsBBM1/OsPLT6, OsBBM2/OsPLT3, and OsBBM3/OsPLT5 genes were significantly downregulated in yel-hc. Other somatic embryogenesis marker genes, such as OsVP1, OsLEC1/OsHAP3E, and OsLEC2/OsFUS3-like 1, also exhibited lower expression in yel-hc than in the wild-type (Fig. 6). These results coincide with the functional characterization of the genes, which demonstrated that triple knockout of the three rice BBM genes (OsBBM1, OsBBM2, and OsBBM3) leads to embryo arrest and abortion (Khanday et al. 2019), and the knockout mutation of OsLEC1/OsNF-YB7 caused abnormal embryo development and embryonic lethality in rice (Niu et al. 2021). Additionally, Khanday et al. demonstrated that OsBBM1 directly regulates the expression of auxin biosynthesis YUCCA genes (OsYUC6, OsYUC7, and OsYUC9), and the formation of embryogenic calli was severely reduced in the Osyuc7 Osyuc9 double mutant and Osyuc6 Osyuc7 Osyuc9 triple mutant rice seeds (Khanday et al. 2020). In the present study, the expression of OsYUC7 and OsYUC9 was significantly downregulated in yel-hc. In our previous study, we tried to generate cop1 loss-of-function plants through anther culture; however, no homozygous recessive plants could be recovered from 293 doubled-haploid plants derived by anther culture (data not shown). This result suggests that recessive mutation of OsCOP1 is deleterious and hinders callus formation. Taken together, these results indicate that OsCOP1 is responsible for somatic embryogenesis in rice, and that its function is intertwined with embryo development and embryonic lethality in plants. In addition, a class of potential flavonoids, such as flavone, flavanone, and flavonol, and their corresponding genes responsible for somatic embryogenesis were identified in cotton, implying that the biosynthesis and accumulation of flavonoids might contribute to transdifferentiation during somatic embryogenesis (Guo et al. 2019). Evidence has also been found for the interaction of microRNA with its target gene (GhmiR157a-GhSPL10), which regulates cellular dedifferentiation and callus proliferation by modulating ethylene-mediated flavonoid biosynthesis during somatic embryogenesis in cotton (Wang et al. 2018). However, the signaling between flavonoid biosynthesis and somatic embryogenesis remains unclear. Thus, further research is needed to understand the regulatory or biochemical mechanism underlying somatic embryogenesis at the molecular level.

Several studies have shown that HY5, a bZIP TF, positively regulates anthocyanin biosynthesis by directly or indirectly regulating the transcription of genes involved in both early and late biosynthetic steps (Maier et al. 2013; Shin et al. 2013; An et al. 2017). In addition, it was reported that HY5 is required for the transcriptional activation of MYB12 and MYB111 genes, which activate FLS under UV-B, to produce flavonol glycosides in Arabidopsis (Stracke et al. 2010). In the present study, the transcription of OsHY5 (LOC_Os06g39960) and OsHY5-like (LOC_Os02g10860) was dramatically increased in developing yel-hc seeds (Table S9). The yel-hc phenotype caused by the mutation of OsCOP1, which inhibits HY5 ubiquitination, supports the regulatory role of COP1–HY5 interaction that COP1 negatively regulates HY5, a positive regulator of flavonoid biosynthesis, resulting in embryo and pericarp coloration in rice seed. In other words, upregulation of HY5 results in the activation of MYB genes, such as OsP1, which regulates flavonoid biosynthesis and subsequently regulates downstream genes to convert the light signal into flavonoids.

COP1 and UV RESISTANCE LOCUS 8 (UVR8) are central regulators of UV-B-induced photomorphogenesis and stress (Favory et al. 2009). The UVR8 monomer, generated by UV-B, directly interacts with the COP1–SPA1 complex, which is separated from the CULLIN 4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 ligase complex and disrupts the COP1-mediated ubiquitination and degradation of HY5 (Huang et al. 2013). The activated UVR8-COP1-HY5 signaling pathway induces the expression of genes encoding two WD40-repeat proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, enabling negative feedback regulation by binding to the UVR8 monomer and mediating UVR8 redimerization in the UVR8-mediated signal transduction pathway (Gruber et al. 2010). In our study, the OsRUP2 (LOC_Os02g02380) gene was highly upregulated in developing yel-hc seeds (Fig. 4). Consistent with the findings in Arabidopsis, it seems that the expression of RUP2 is mediated by COP1 in rice and the RUP2 protein directly interacts with UVR8-COP1. In addition, COP1 is able to target RUP proteins for ubiquitination and degradation, resulting in the regulation of RUP accumulation and the stabilization of HY5 proteins (Ren et al. 2019; Gruber et al. 2010). Besides UV-B-induced photomorphogenesis, flavonoid and anthocyanin biosynthesis pathways are closely related to the UV-B signaling pathway (Jaakola 2013; Peng et al. 2013); therefore, it is important to further study the biological function of RUP proteins and their interaction with COP1 and HY5 in rice.

Conclusion

In this study, transcriptome profiling was conducted to identify key genes involved in COP1-mediated flavonoid (C-glycosyl flavone) biosynthesis and embryogenesis in the developing seeds of yel, cop1 null mutant in rice, and to elucidate the underlying molecular mechanisms. Genes involved in C-glycosyl flavone biosynthesis were significantly upregulated and potentially play a crucial role in the pigmentation of embryo and pericarp in yel-hc. Mutation in OsCOP1 reduced the expression level of genes responsible for somatic embryogenesis as well as zygotic embryogenesis. The identified putative genes that govern flavonoid biosynthesis and embryogenesis could be used to increase the content of the medicinally beneficial C-glycosyl flavones in rice seeds through metabolic engineering approaches and provide pivotal information on embryo formation and development.