Identification of the UGT Family and Functional Validation of MwUGT2 in Meconopsis wilsonii

Abstract

Flower color is one of the most ornamental values of Meconopsis wilsonii, but very limited studies have been reported on its flower color formation. The UDP-glycosyltransferase (UGT) gene family plays a crucial role in plant flower color formation. In this study, the full-length transcriptome data of M. wilsonii was used to identify MwUGTs, focusing on protein physicochemical properties' subcellular localization, and phylogenetic relationships. In addition, sequence analysis, expression pattern analysis, subcellular localization, and functional validation of MwUGT2 were also performed. A total of 26 MwUGTs were identified in full-length transcriptome and clustered into eight subgroups. Phylogenetic analysis and KEGG database annotation showed that MwUGT2 is associated with anthocyanin synthesis and accumulation. Subsequently, based on the expression of MwUGT2 during flower development and in different tissues, it was preliminarily determined that MwUGT2 plays a role in the flower bud stage. Subcellular localization assays suggested that MwUGT2 is present in the nucleus and cytoplasm. Overexpression in Nicotiana tabacum showed that MwUGT2 significantly increased the content of Cyanidin-3-O-glucoside and resulted in dark pink flowers in transgenic plants. In summary, our findings suggest that MwUGT2 plays a crucial role in the biosynthesis of anthocyanin and will also contribute to understanding the mechanisms of flower color formation in M. wilsonii.

Keywords: Cyanidin-3-O-glucoside; Cyanidin-3-O-sambubioside; flower color; metabolomics; transgenic tobacco.

Figure 1

Phylogenetic analysis of the MwUGT proteins among M. wilsonii and A. thaliana. Different colored strips indicate subfamilies. MwUGTs were divided into A, B, D, E, F, G, J, and L by the known AtUGT proteins.

Figure 2

Phylogeny tree and sequence alignment of UGTs from different species. (A) Phylogenetic analysis of UGT proteins from different species. GenBank accession numbers are as follows: QrUGT (Quercus robur XP_050253999.1), QsUGT (Quercus suber XP_023873100.1), QlUGT (Quercus lobata XP_030934873.1), PaUGT (Populus alba XP_034907570.1), PtUGT (Populus trichocarpa XP_006376354.2), PdUGT (Paeonia delavayi AQZ26785.1), VlUGT (Vitis labrusca ABR24135.1), CfUGT (Cornus florida XP_059636915.1), AcUGT (Aralia cordata BAD06514.1), AgUGT (Apium graveolens AXU98426.1), PsUGT (Papaver somniferum XP_026387438.1), NnUGT (Nelumbo nucifera XP_010279580.1), TsUGT (Telopea speciosissima XP_043695717.1) (B) Amino acid sequences alignment of MwUGT2 protein in M. wilsonii with proteins from other species.

Figure 3

The expression pattern and subcellular localization of MwUGT2. (A) Phenotypes of the three stages of the flowering process and different tissues. (B) Relative expression levels of MwUGT2 gene in the three stages of the flowering process and different tissues, including bud stage, dehiscence stage, full-spread stage, root stem, and leaf. The expression levels of MwUGTw2 in the full-spread stage have been arbitrarily set = 1. Error bars indicate standard deviations and different letters above the bars represent significant differences (p < 0.05) according to Duncan’s statistical analysis. (C) Subcellular localization of MwUGT2-GFP heterologously expressed in Nicotiana tabacum leaves. Scale bar, 20 μm.

Figure 4

Overexpression of MwUGT2 contributes to petal discoloration in transgenic tobacco lines. (A) Tobacco flowers of wide-type and transgenic lines. WT, wild-type; OE, overexpression. (B) Expression profiles of MwUGT2 in transgenic tobacco flowers. (C) The expression of MwUGT2 in transgenic lines and WT. The expression level of MwUGT2 in WT has been arbitrarily set = 1. Error bars indicate standard deviations and different letters above the bars represent significant differences (p < 0.05) according to Duncan’s statistical analysis, (D) Analogous. (D) The expression patterns of NtCHS, NtCHI, NtF3H, NtF3′5′H, NtDFR, NtANS and NtUFGT in WT and transgenic plants. The expression level of NtCHS, NtCHI, NtF3H, NtF3′5′H, NtDFR, NtANS and NtUFGT in WT have been arbitrary set = 1, respectively.

Figure 5

Effect of MwUGT2 on anthocyanin accumulation in transgenic tobacco flower. (A) Quantity of anthocyanin metabolites in WT and transgenic tobacco petals. (B) The content of Pelargonidin, Cyanidin, Peonidin, Delphinidin, Petunidin, Malvidin, Procyanidin, and Flavonoid in flowers of WT and transgenic plants. Statistical significance was determined using Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns indicates not significant). (C) Heat map of anthocyanin differential metabolites.

Figure 6

M. wilsonii metal ions and pH. (A) Metal ion content in different developmental stages and different tissue parts of M. wilsonii flowers. Error bars indicate standard deviations and different letters above the bars represent significant differences (p < 0.05) according to Duncan’s statistical analysis, Figure 6B Analogous. (B) pH values for different developmental stages of M. wilsonii flowers.

Figure 7

Schematic diagram of the formation of blue-violet flowers of M. wilsonii.