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. 2023 Jun 21:14:1186816.
doi: 10.3389/fpls.2023.1186816. eCollection 2023.

Enhancement of the anthocyanin contents of Caladium leaves and petioles via metabolic engineering with co-overexpression of AtPAP1 and ZmLc transcription factors

Affiliations

Enhancement of the anthocyanin contents of Caladium leaves and petioles via metabolic engineering with co-overexpression of AtPAP1 and ZmLc transcription factors

Ximeng Yang et al. Front Plant Sci. .

Abstract

Introduction: Metabolic engineering of anthocyanin synthesis is an active research area for pigment breeding and remains a research hotspot involving AtPAP1 and ZmLc transcription factors. Caladium bicolor is a desirable anthocyanin metabolic engineering receptor, with its abundant leaf color and stable genetic transformation system.

Methods: We transformed C. bicolor with AtPAP1 and ZmLc and successfully obtained transgenic plants. We then used a combination of metabolome, transcriptome, WGCNA and PPI co-expression analyses to identify differentially expressed anthocyanin components and transcripts between wild-type and transgenic lines.

Results: Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and peonidin-3-O-rutinoside are the main components of anthocyanins in the leaves and petioles of C. bicolor. Exogenous introduction of AtPAP1 and ZmLc resulted in significant changes in pelargonidins, particularly pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside in C. bicolor. Furthermore, 5 MYB-TFs, 9 structural genes, and 5 transporters were found to be closely associated with anthocyanin synthesis and transport in C. bicolor.

Discussion: In this study, a network regulatory model of AtPAP1 and ZmLc in the regulation of anthocyanin biosynthesis and transport in C. bicolor was proposed, which provides insights into the color formation mechanisms of C. bicolor, and lays a foundation for the precise regulation of anthocyanin metabolism and biosynthesis for economic plant pigment breeding.

Keywords: AtPAP1; Caladium bicolor; ZmLc; anthocyanin biosynthesis; metabolic engineering.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Phenotype and differentially accumulated metabolites analysis of metabolome in TTs and WTs. (A), The phenotype of callus, roots, petioles, leaves of transgenic lines and WT. T1-T4, representing four growth periods of C. bicolor. (B), Classification and statistical analysis of all metabolites detected. (C), The relative content of anthocyanins. (D), Number of DMs among all comparison units. (E), Venn analysis among WT2L vs. TT2L, WT2P vs. TT2P, WT3L vs. TT3L and WT3P vs. TT3P. (F), The heatmap analysis of overlapping DMs according to the relative content in samples. (G), The relative content of overlapping different anthocyanins.
Figure 2
Figure 2
Differentially expressed unigenes (DEGs) in TTs and WTs. (A), The number of up- and down-regulated genes in WT2L vs. TT2L, WT2S vs. TT2S, WT3L vs. TT3L, WT3S vs. TT3S, and WT2L vs. WT3L. (B), GO enrichment analysis. (C), KEGG enrichment analysis.
Figure 3
Figure 3
Weighted gene co-expression network analysis. (A), Hierarchical clustering tree (cluster dendrogram) results showed 11 expression modules, labeled with different colors. (B), Module correlations and corresponding p-values. The left panel shows the 11 modules and the number of genes in each module. The value inside each box represents Pearson’ s correlation coefficient between the module with anthocyanin, and the number in each parentheses represents p-value. The color scale on the right represents the degree of correlation between modules and anthocyanins and the red represent high correlation. (C), KEGG enrichment analysis of genes in ‘blue’ module. (D), KEGG enrichment analysis of genes in ‘pink’ module. (E), The heatmap of identified TFs in ‘blue’ and ‘pink’ modules.
Figure 4
Figure 4
The identification of DEGs in ‘blue’ and ‘pink’ modules of anthocyanin biosynthesis and transport pathway.
Figure 5
Figure 5
Connection network between KEY DEGs and differential metabolites. (A), Gene co-expression network of key genes by WGCNA analysis. (B), Co-expression network of key genes and anthocyanins content by Pearson analysis. (C), Gene co-expression network of key genes by PPI analysis. The circle sizes represent the number of connections between genes. The red, blue and green circles represent transcription factors, structural genes of anthocyanin biosynthesis and transporters, respectively. (D), Phylogenetic relationships of MYB-TFs. The MYBs belonging to the SG4-SG6 and R3-MYB were used to build a phylogenetic tree based on maximum likelihood, which was employed to infer the MYB candidates.
Figure 6
Figure 6
Validation of the expression of pigment-related genes in C. bicolor by qRT-PCR.

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