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. 2023 Feb 23;14(3):559.
doi: 10.3390/genes14030559.

LhANS-rr1, LhDFR, and LhMYB114 Regulate Anthocyanin Biosynthesis in Flower Buds of Lilium 'Siberia'

Shaozhong Fang  1 , Mi Lin  1 , Muhammad Moaaz Ali  2 , Yiping Zheng  1 , Xiaoyan Yi  2 , Shaojuan Wang  2 , Faxing Chen  2 , Zhimin Lin  1
Affiliations

LhANS-rr1, LhDFR, and LhMYB114 Regulate Anthocyanin Biosynthesis in Flower Buds of Lilium 'Siberia'

Shaozhong Fang et al. Genes (Basel). .

Abstract

The bulb formation of Lilium is affected by many physiological and biochemical phenomena, including flower bud differentiation, starch and sucrose accumulation, photoperiod, carbon fixation, plant hormone transduction, etc. The transcriptome analysis of flower buds of Lilium hybrid 'Siberia' at different maturity stages showed that floral bud formation is associated with the accumulation of anthocyanins. The results of HPLC-MS showed that cyanidin is the major anthocyanin found in Lilium 'Siberia'. Transcriptome KEGG enrichment analysis and qRT-PCR validation showed that two genes related to flavonoid biosynthesis (LhANS-rr1 and LhDFR) were significantly up-regulated. The functional analysis of differential genes revealed that LhMYB114 was directly related to anthocyanin accumulation among 19 MYB transcription factors. Furthermore, the qRT-PCR results suggested that their expression patterns were very similar at different developmental stages of the lily bulbs. Virus-induced gene silencing (VIGS) revealed that down-regulation of LhANS-rr1, LhDFR, and LhMYB114 could directly lead to a decrease in anthocyanin accumulation, turning the purple phenotype into a white color. Moreover, this is the first report to reveal that LhMYB114 can regulate anthocyanin accumulation at the mature stage of lily bulbs. The accumulation of anthocyanins is an important sign of lily maturity. Therefore, these findings have laid a solid theoretical foundation for further discussion on lily bulb development in the future.

Keywords: Lilium ‘Siberia’; MYB transcription factor; VIGS; anthocyanin; bulb; full-length transcriptomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The appearance and morphology of Lilium bulb and bud at different developmental stages (A–F). (A) The bulb diameter is 3 cm; (B) the bulb diameter is 6 cm; (C) the bulb diameter is 10 cm; (D) the bulb diameter is 14 cm; (E) the bulb diameter is 16 cm; (F) the bulb diameter is 22 cm. The data represent the average of three biological replicates.
Figure 2
Figure 2
The concentrations of anthocyanin-related compounds in the flower buds of oriental lily ‘Siberia’. High-performance liquid chromatography-mass spectrometry (HPLC-MS) chromatograms suggested the presence of anthocyanin aglycones was completed at 530nm; the compounds were identified by comparing with the mass spectra of the standards. A—delphinidin, B/B1—cyanidin, C—petunidin, D—pelargonin, E—mallow pigment. Black color represents the standard, and red or green represents the text sample.
Figure 3
Figure 3
Transcriptome analysis and qRT-PCR validation of the candidate DEGs involved in anthocyanin biosynthesis in Lilium ′Siberia′. (A) A heatmap showing the expression patterns of the DEGs involved in anthocyanin biosynthesis. The heatmap was generated based on the normalized log2 (FoldChange) > 1 values for each DEG. Green and red scales represent relatively low or high expression, respectively. (B) Schematic diagram of metabolic pathway for cyanidin. (C) Volcano plot of the total differentially expressed genes in both E and F groups using the threshold of p < 0.05 and |log2FoldChange| > 1. The x-axis represents the log2 (fold change) values for gene expression, and the y-axis represents the −log10 (p value). (D) The qRT–PCR validation of the DEGs involved in anthocyanin biosynthesis. Expression data are the represent as the mean values of three biological replicates ± SD. Yellow color represents significant up-regulation of genes, while blue color represents insignificant changes.
Figure 4
Figure 4
Genetic expressions of MYB genes and cluster analysis in transcriptome data. (A) The numbers and families of top 28 TFs enriched in flower bud. (B) The heatmap analysis of MYB DEGs through transcriptome data. The heatmap was generated based on the normalized log2 (FoldChange) > 1 values for each DEG. The color scale at the right represents gene expression values (the red corresponds to genes with high expression and the green corresponds to genes with low expression). (C) The cluster analysis map of R2R3-MYB proteins. The cluster analysis tree was completed using MEGA 5.0 software with the Arabidopsis MYB genes as reference. The aim gene of LhMYB114 is highlighted in red color.
Figure 5
Figure 5
Amino acid sequence alignment of LhMYB114. (A) The phylogenetic analysis of LhMYB114 with homologous genes related to anthocyanin biosynthesis. (B) The important domain analysis of LhMYB114. Color represents amino acid sequence is high similarity, including pink, blue, cyan.
Figure 6
Figure 6
The qRT–PCR analysis of 3 key genes responsible for anthocyanin metabolism in flower buds of lily at different development stages. (A) The expression of LhMYB114 genes at different stages. (B) The expression of LhDFR genes at different stages. (C) The expression of LhANS-rr1 genes at different stages. The values are means ± SDs (n = 6). The data were normalized to a value of 1 for the A group. Significant differences were determined by Student′s t test (** p < 0.01; *** p < 0.001).
Figure 7
Figure 7
Transient silencing phenotypes among LhMYB114, LhANS-rr1, and LhDFR in Lilium flower buds by virus-induced gene silencing (VIGS). (A) Photographs of Lilium flower buds from TRV-infected plants. (B) RT-PCR detection of RNA1 and RNA2 of TRV1 and TRV2 in Lilium.
Figure 8
Figure 8
Relative expression levels of three key genes under different VIGS treatments. (A) Down-regulation of LhANS-rr1 and LhDFR expression in LhMYB114-silenced bud. (B) Down-regulation of LhMYB114 and LhDFR expression in LhANS-rr1-silenced bud. (C) Down-regulation of LhANS-rr1 and LhMYB114 expression in LhDFR-silenced bud. (D) Down-regulation of LhMYB114, LhANS-rr1 and LhDFR expression in LhMYB114+LhANS-rr1-silenced bud. (E) Down-regulation of LhMYB114, LhANS-rr1 and LhDFR expression in LhMYB114+LhDFR-silenced bud. (F) Down-regulation of LhMYB114, LhANS-rr1 and LhDFR expression in LhMYB114+LhDFR+LhANS-rr1-silenced bud. (G) The six main genes (C4H, 4CL, CHS, CHI, F3H, and UFGT) expressed in TRV-LhMYB114, TRV-LhANS-rr1, TRV-LhDFR, and empty vector. The values are means ± SDs (n = 3). Lowercase letters (a,b) indicate statistically significant differences at p < 0.05.
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