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. 2023 Jan 17;24(3):1844.
doi: 10.3390/ijms24031844.

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Spot Formation on Lilium leichtlinii var. maximowiczii Flower Petals

Affiliations

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Spot Formation on Lilium leichtlinii var. maximowiczii Flower Petals

Zhen Wang et al. Int J Mol Sci. .

Abstract

Plants exhibit remarkable diversity in their petal colors through biosynthesis and the accumulation of various pigments. Lilium, an important cut and potted flower, has many coloring pattern variations, including bicolors and spots. To elucidate the mechanisms regulating spot formation in Lilium leichtlinii var. maximowiczii petals, we used multiple approaches to investigate the changes in petal carotenoids, spot anthocyanins, and gene expression dynamics. This included green petals without spots (D1-Pe and D1-Sp), yellow-green petals with purple spots (D2-Pe and D2-Sp), light-orange petals with dark-purple spots (D3-Pe and D3-Sp), and orange petals with dark-purple spots (D4-Pe and D4-Sp). D3-Pe and D4-Pe contained large amounts of capsanthin and capsorubin and small amounts of zeaxanthin and violaxanthin, which contributed to the orange color. In addition to cyanidin-3-O-glucoside, pelargonidin-3-O-rutinoside, cyanidin-3-O-rutinoside, and peonidin-3-O-rutinoside may also contribute to L. leichtlinii var. maximowiczii's petal spot colors. KEGs involved in flavonoid biosyntheses, such as CHS, DFR, and MYB12, were significantly upregulated in D2-Sp and D3-Sp, compared with D1-Sp, as well as in spots, compared with petals. Upregulated anthocyanin concentrations and biosynthesis-related genes promoted spot formation and color transition. Our results provide global insight into pigment accumulation and the regulatory mechanisms underlying spot formation during flower development in L. leichtlinii var. maximowiczii.

Keywords: Lilium leichtlinii var. maximowiczii; anthocyanin biosynthesis; metabolomics; petal spots; transcriptomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphological observation of L. leichtlinii var. maximowiczii flowers. (A) Morphological anatomy of L. leichtlinii var. maximowiczii flowers during floral development. Development stages D1 (green petals without spots), D2 (yellowish green petals with purple spots), D3 (light-orange petals with dark-purple spots), D4 (orange petals with dark-purple spots, one day before anthesis), and D5 (the day of anthesis). Bar = 1 cm. (B) Sampling strategy: petals and spots were collected at D1, D2, D3, and D4 for transcriptome sequencing and metabolome identification. (C) Scanning electron microscopy appearance of raised spots on D2 petals. Bar = 500 µm.
Figure 2
Figure 2
The anthocyanin content detected in different samples in this study. The x-axis represents the anthocyanin levels (µg/g). The y-axis represents the anthocyanin composition obtained using high-performance liquid chromatography (HPLC). Error bars show the SD of the mean. D1-Sp, lower part of green petals at stage 1; D2- Sp, D3- Sp, D4- Sp, spots on petals at different stages.
Figure 3
Figure 3
Heatmap of metabolites related to cyanidin, delphinidin, flavonoid, malvidin, pelargonidin, peonidin, and petunidin in D1-Sp, D2-Sp, D3-Sp, and D4-Sp. The marker on the right side of the heatmap represents the name of each anthocyanin composition obtained via HPLC.
Figure 4
Figure 4
Differentially accumulated metabolite (DAM) analysis of the metabolome. Heatmaps of DAMs in D1-Sp vs. D2-Sp (A), D1-Sp vs. D3-Sp (B), and D1-Sp vs. D4-Sp (C). (D) Venn analysis of D1-Sp vs. D2-Sp, D1-Sp vs. D3-Sp, and D1-Sp vs. D4-Sp. The color scale from Min (blue) to Max (red) indicates the metabolite contents from low to high. Identification of DAMs between three comparison groups was performed using variable importance in projection values ≥1 and fold change ≥2 or ≤0.5; in addition, the content must be ≥ 1 μg/g DW in spots in at least one stage.
Figure 5
Figure 5
Gene regulation during spot formation from D1 to D4. (A) K-means cluster analysis of co-expressed genes and their expression patterns. (B) Differentially expressed genes (DEGs) involved in transcription factor enrichment in the light yellow module. (C) KEGG enrichment bar plot of DEGs in the light yellow module. The light yellow cluster represents the expression pattern of 1618 co-expressed genes identified via K-means cluster analysis. (D) KEGG enrichment bar plot of DEGs in the dark magenta module including 318 identified co-expressed genes.
Figure 6
Figure 6
Analysis of DEGs in the anthocyanin biosynthesis pathway in spots on L. leichtlinii var. maximowiczii petals. (A) Reconstruction of the anthocyanin biosynthetic pathway with the structural DEGs. The DEGs were identified using an adjusted p value < 0.05 and |log2 fold change| ≥ 1. (B) Validation of the expression of anthocyanin-related genes in Pe and Sp at the four stages using RT-qPCR. Error bars indicate the SD of three independent biological repeats.

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