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A determination of anthocyanin content was performed using three-period petals in S1 (bud stage), S2 (coloring stage), and S3 (blooming stage) to measure the pigmentation in lily petals during development. Each sample contained three replicates of different triennial plants. The color of the S1 petal samples was relatively light compared to the other two samples (Fig. 1a). This parallels the total anthocyanin content measured by anthocyanin content extracted, which was lower in the S1 sample compared to S3 (Fig. 1b). The anthocyanins content in the petals were lower in S1 and higher in S3. The content trend of each cyanidin derivative, including cyanidin, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside, was the same as that of the total anthocyanins (Fig. 1c). Overall, the content of total anthocyanins and their components in lily petals gradually accumulated with the development of the petals.
Figure 1.
Determination of anthocyanin content in lily petals. (a) Lily petals in different periods. (b) Anthocyanin content in lily petals at different periods of lily development. (c) The relative content of cyanidin and its compounds in petals determined in metabolomics analysis. * Denotes statistically significant differences between samples. * p-value ≤ 0.1; ** p-value ≤ 0.05; *** p-value ≤ 0.01.
miRNA sequencing and the target genes prediction
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miRNA sequencing was carried out on lily flower petals to investigate the genes involved in color creation. We constructed a miRNA library and 10 M clean data generated from sequencing. The Rfam database was selected to annotate and sequence small RNA sequences. Subsequently, unique sequences were analyzed by miRBase 22.0 and RNAfold software to identify the known and novel miRNAs. Finally, it was mapped to the corresponding lily transcriptome using UniGene. A total of 326 miRNAs were obtained through miRNA sequencing from nine libraries, including 285 known miRNAs and 41 new miRNAs. The 285 known miRNAs belonged to 41 families (Fig. 2a). The miR396, miR166, miR156, etc. gene families had the most members.
Figure 2.
The classification of miRNA and the target genes. (a) The selected 285 known miRNA belonged to 41 MiRNA families. (b) Heatmap of DEMs expression in the development of flower. After performing row clusters, each colored cell shows the average log2 (FPKM) value of each miRNA. (c) DEMs in floral development is depicted in a Venn diagram.
To further understand the role of miRNA in the process of flower color formation, we used psRNATarget to predict the miRNA target genes from the data of RNA-seq unigenes and found that 193 known miRNAs targeted 1,374 transcripts. There were 75 significant differentially expressed miRNAs (DEMs) selected by the threshold of a fold change > 2 with a p-value < 0.05 in all the compared groups (Fig. 2b). A total of 12 DEMs (six up, six down) in S2 vs S1; 58 DEMs (32 up, 26 down) in S3 vs S1; 29 DEMs (26 up, 3 down) in S2 vs S3 (Fig. 2c).
According to the psRNATarget prediction, a total of 898 potential target genes were identified in lily flower petals, which were targeted by the 75 DEMs. GO analysis and KEGG annotation were performed to evaluate the potential functions of these miRNA target genes. The GO analysis showed that these target genes were divided into three categories: cellular components, molecular functions, and biological processes (Supplemental Fig. S1a). In biological processes, the oxidation-reduction process was one of the biggest groups. In cellular components, the integral component of the membrane was the major group. Molecular functions concentrated on zinc ion binding, DNA binding, and ATP binding. KEGG analysis showed that the significantly enriched pathways of the target genes were in metabolism progress (Supplemental Fig. S1b), indicating that target genes regulated by miRNA would have a certain influence on plant metabolic activities. In addition, some target genes were enriched in flavone and flavonol biosynthesis, suggesting that miRNA may indirectly affect the flavonoid biosynthesis pathway.
Screening and verification of miRNA target genes related to petal color in lilies
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We further analyzed the annotated target genes and found among them many genes encoding transcription factors. The expression levels were similar to the accumulation trend of total anthocyanins and cyanidin derivatives (Fig. 1b, c), and opposite to the expression levels of the corresponding miRNA (Fig. 3a, b), indicating that miRNA negatively regulates the expression of target genes. Therefore, we analyzed the correlation of expression trend between miRNA and the target genes. Gene pairs with p-value ≤ −0.5 were selected for the study data. A correlation network diagram was then created using the Cytoscape program (Fig. 3c).
Figure 3.
The correlation between miRNA and target genes screened from lily petals related to anthocyanin synthesis. (a) Heatmap of miRNA expression. (b) MiRNA target gene expression heatmap. The average log2 (FPKM) value of each gene is shown by the color of each cell. (c) The network of target genes and miRNAs controls the progress of anthocyanins synthesis. TF genes are represented by the blue rhombic nodes, miRNA are represented by the purple oval nodes; black lines represent negative correlation; and blue lines represent positive correlation. (d) The Sankey plot of miRNA and target genes.
Finally, we screened the target genes including SPL (SQUAMOSA promoter-binding protein-like), MYB, WD, 3GT (anthocyanidin 3-O-glucosyltransferase), GRF (growth regulation factor), ARF (Auxin response factor), NAC,and MADS, which were targeted by miR156, miR828, miR166, miR396, miR160, miR167, miR164, and miR5179, respectively (Table 1). The regulation model of these miRNAs and target genes can be divided into two categories: one miRNA target regulates multiple gene transcripts, or several miRNAs target regulate the same gene transcript (Fig. 3d). We found that, miR159 can target multiple SPL, miR164 can target multiple NAC, miR396 can target 3GT and GRF, and both miR160 and miR167 can target ARF, miR828, miR166, miR5179 can target MYB, WD, MADS, respectively (Table 1). In future research, we will select these miRNAs and their target genes for further study.
Table 1. Target genes of anthocyanin biosynthesis related miRNAs in lily petals.
miRNA family miRNAs Target genes Target gene annotation miR156 osa-miR156a_L+1 TRINITY_DN91060_c0_g1, TRINITY_DN95303_c1_g1, TRINITY_DN91754_c0_g1, TRINITY_DN95303_c2_g1, TRINITY_DN94725_c0_g1, TRINITY_DN102736_c2_g5 SPL miR828 cme-miR828 TRINITY_DN103447_c0_g1 R2R3-MYB miR166 osa-miR166a-5p_1ss7TC TRINITY_DN101304_c1_g1 WD miR396 osa-miR396a-5p_L+1 TRINITY_DN101276_c1_g1 3GT csi-miR396f-5p_1ss20TC, osa-miR396c-5p_R-1, osa-miR396a-5p_R+1, osa-miR396a-5p_1ss21GA, osa-miR396a-5p TRINITY_DN92078_c0_g1 GRF csi-miR396f-5p_1ss20TC, osa-miR396c-5p_2ss7AG21TA, osa-miR396c-5p_R-1, osa-miR396a-5p_R+1, osa-miR396a-5p_1ss21GA, osa-miR396a-5p, osa-miR396a-5p_L+1, csi-miR396a-5p_R+2_1ss7AG TRINITY_DN88167_c0_g1 GRF osa-miR396c-5p_R-1, osa-miR396a-5p_R+1, osa-miR396a-5p_1ss21GA, osa-miR396a-5p, csi-miR396f-5p_1ss20TC, osa-miR396a-5p_L+1,
csi-miR396a-5p_R+2_1ss7AGTRINITY_DN94882_c0_g1 GRF csi-miR396f-5p_1ss20TC, osa-miR396c-5p_R-1, osa-miR396a-5p_1ss21GA, osa-miR396a-5p, osa-miR396a-5p_L+1, csi-miR396a-5p_R+2_1ss7AG TRINITY_DN88510_c0_g1 GRF miR5179 osa-miR5179, osa-miR5179_R+2 TRINITY_DN96747_c12_g4 MADS miR164 osa-miR164a, osa-miR164a_R+1 TRINITY_DN96598_c0_g1 NAC osa-miR164a_R+1, osa-miR164a, aof-miR164_R+2 TRINITY_DN96199_c2_g2 NAC miR160 osa-miR160a-5p_R-1_1ss20CT TRINITY_DN100728_c0_g1 ARF miR167 osa-miR167d-5p_R+2 TRINITY_DN99246_c0_g1 ARF bdi-miR167c-5p_L+1R-2, osa-miR167d-5p_R+2 TRINITY_DN99246_c0_g3 ARF To reveal the expression patterns of anthocyanin biosynthesis-related miRNAs in lily petals, nine miRNAs and 11 unigenes were selected for RT-qPCR validation (Fig. 4). Generally, the expression patterns of miRNA and unigenes were consistent with the results of the high throughput sequencing, indicating that the sequencing data were reliable. In the process of anthocyanin synthesis in lily petals, LvmiR164 (osa-miR164a_R+1) and LvmiR166 (osa-miR166a-5p_1ss7TC) showed down-regulated pattern, and LvmiR160 (osa-miR160a-5p_R-1_1ss20CT), LvmiR167c (bdi-miR167c-5p_L+1R-2), LvmiR5179 (osa-miR5179_R+2), LvmiR828 (cme-miR828), LvmiR156 (osa-miR156a_L+1) and LvmiR396 (osa-miR396a-5p_L+1) showed up-regulated pattern (Fig. 4). RT-qPCR was also used to confirm the target genes' corresponding expression patterns, including LvNAC1 (TRINITY_DN96598_c0_g1) and LvNAC2 (TRINITY_DN96199_c2_g2) for LvmiR164, LvWD (TRINITY_DN101304_c1_g1) for LvmiR166, LvARF1 (TRINITY_DN100728_c0_g1) and LvARF2 (TRINITY_DN99246_c0_g3) for LvmiR160 and LvmiR167c, LvMADS (TRINITY_DN96747_c12_g4) for LvmiR5179, LvMYB5 (TRINITY_DN103447_c0_g1) for LvmiR828, Lv3GT (TRINITY_DN101276_c1_g1) for LvmiR396, and LvSPL1 (TRINITY_DN91754_c0_g1), LvSPL2 (TRINITY_DN102736_c2_g5), and LvSPL3 (TRINITY_DN95303_c2_g1) for LvmiR156. The expression levels increased in LvNAC1/2 and LvWD and decreased in LvARF1/2, LvMADS, and LvSPL1/2/3. LvMYB5 and, Lv3GT increased in S2 and then decreased slightly in the S3 stage (Fig. 4). In general, the expression trend of the target genes was opposite to that of the miRNA.
Figure 4.
The expression and the regulation of miRNA and the target genes. 2−ΔΔCᴛ method was used to measure genes' relative expression in S1, S2, S3 stage of lily cultivar Vivian. * Denotes statistically significant differences between samples.
Consistent with previous experiments, the content of the anthocyanin substances was lowest in the S1 stage and gradually increased in the S2 and S3 stages. This parallels the expression levels of Lv3GT and LvMYB5, which were perhaps the positive regulation genes related to anthocyanin biosynthesis in the lily petals[37,38]. In contrast, for flower coloring, significant inverse relationships between 3GT and LvMYB5 expression levels and LvmiR396 and LvmiR828 expression levels were found in the S2 and S3 stages. These findings suggest that the expression levels of these target genes and miRNAs are related. miRNAs in the pigmentation of lily flowers negatively regulated their target genes.
Analysis of the targeted regulation of LvMYB5 by miR828
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Lv-miR828 targeted LvMYB5 as analyzed by psRNATarget (Table 1), which is directly related to the anthocyanin synthesis pathway[37,43]. This regulatory pathway is therefore likely to affect lily pigmentation, and so we conducted further analysis on these genes.
From the multi-sequence alignment analysis of mature LvmiR828, we found it shares a high identity with miR828 in other plants, where it functions in anthocyanin biosynthesis. There was only one nucleotide difference between the mature miR828 (Fig. 5a), indicating that LvmiR828 might play a similar role here as in other species. The cleavage site was predicted using psRNATarget to be in the CDS sequence of LvMYB5 and the motif site of LvMYB5, coding for helix 3 of specific R3 domain, was targeted by LvmiR828 (Fig. 5b). We further compared the amino acid sequences of the target gene MYB in Arabidopsis thaliana and grapes with LvMYB5 in lilies, and found that the target sites were located at the end of R3 region with high similarity (Fig. 5c). We therefore speculate that miR828 targets LvMYB5 transcriptional translation through the cleavage site and inhibits anthocyanin synthesis.
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MiRNAs are important post-transcriptional regulators, some of which participate in regulating anthocyanin biosynthesis and other pathways, including plant stress, growth and development, and internal and external hormones[23,24], however, the study of miRNAs involved in lily pigmentation is incomplete. In this study, we comprehensively performed the RNA-seq and miRNA sequencing of lily petals at three stages of flower coloring. A total of 326 miRNAs were identified from nine libraries, including 285 known and 41 new miRNAs, using the Illumina Next Seq 500 platform. The differential miRNAs were screened, and the functions of the target genes were analyzed. The expression patterns of candidate miRNAs and target genes were detected by qPCR to obtain the genes that may function in flower coloration in lily petals. Identification and functional analysis of miRNAs and target genes related to flower coloring will help reveal the coloration regulation mechanism in ornamental plants in complex and variable environments.
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About this article
Cite this article
Yin X, Gao Z, Sun S, Zhang L, Irfan M, et al. 2023. Insights into microRNA regulation of flower coloration in a lily cultivar Vivian petal. Ornamental Plant Research 3:9 doi: 10.48130/OPR-2023-0009
Insights into microRNA regulation of flower coloration in a lily cultivar Vivian petal
- Received: 28 September 2022
- Accepted: 06 March 2023
- Published online: 24 April 2023
Abstract: MicroRNAs (miRNAs) are a class of non-coding small RNAs involved in the negative regulation of gene expression, which plays critical roles in developmental and metabolic pathways. However, it is not well understood how miRNA regulate the anthocyanin biosynthesis pathway in lily flowers. Using miRNA sequencing and target gene expression analysis, we explored the regulatory networks of miRNAs and their target-related flower coloration in lily petals. A total of 326 miRNAs were obtained by miRNA sequencing, including 285 known miRNAs and 41 new miRNAs. According to the psRNATarget prediction, there were a total of 75 differentially expressed miRNAs (DEMs) that target 898 potential genes. We also screened the target genes including LvSPL, LvMYB5, LvWD, Lv3GT, LvGRF, LvARF, LvNAC, and LvMADS, which were targeted by LvmiR156, LvmiR828, LvmiR166, LvmiR396, LvmiR160, LvmiR167, LvmiR164, and LvmiR5179. These genes may be involved in regulating other secondary metabolic pathways, and forming a complex regulatory network of anthocyanin biosynthesis. We therefore proposed a putative miRNA-target module associated with anthocyanin biosynthesis. In addition, we predicted the binding site of LvMYB5, the target gene of miR828, and speculated that miR828 targets regulate LvMYB5 transcriptional translation through a cleavage site, which then inhibits anthocyanin synthesis. Our findings contribute to an understanding of the functional characterization of miRNAs and their targets in controlling anthocyanin production in plants and may lead to future identification and characterization of miRNAs in lilies.
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Key words:
- MicroRNA /
- Regulations /
- Coloration /
- Lily