Two responses to MeJA induction of R2R3-MYB transcription factors regulate flavonoid accumulation in Glycyrrhiza uralensis Fisch

Abstract

Flavonoids are key components of licorice plant that directly affect its medicinal quality. Importantly, the MYB family of transcription factors serves to regulate the synthesis of flavonoids in plants. The MYB transcription factors represent one of the largest families of transcription factors in plants and play important roles in the process of plant growth and development. MYB gene expression is induced by a number of plant hormones, including the lipid-based hormone jasmonate (JA). Methyl jasmonate (MeJA) is an endogenous plant growth regulator that can induce the JA signaling pathway, which functions to regulate the synthesis of secondary metabolites, including flavonoids. In this study, MeJA was added to licorice cell suspensions, and RNA-seq analysis was performed to identify the differentially expressed genes. As a result, the MYB transcription factors GlMYB4 and GlMYB88 were demonstrated to respond significantly to MeJA induction. Subsequently, the GlMYB4 and GlMYB88 protein were shown to localize to the cell nucleus, and it was verified that GlMYB4 and GlMYB88 could positively regulate the synthesis of flavonoids in licorice cells. Overall, this research helps illustrate the molecular regulation of licorice flavonoid biosynthesis induced by MeJA.

Fig 1. Expression analysis of the CHS and C4H genes in licorice cells.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for the untreated cell: *P < 0.05. The expression levels of CHS and C4H in cells treated with MeJA at different times as based on qPCR analysis; C4H: cinnamic acid-4-hydroxylase; CHS: chalcone synthase.

Fig 2. Sequence data analysis.

(A) Enriched GO Terms. Find out which of the different genes are higher in the GO group. (B) KEGG pathway enrichment diagram depicting the differentially expressed genes (DEGs) among the metabolic pathways. (C) Classification of the genes with significant differential expression. Identity of the genes with the most apparent changes in their expression levels.

Fig 3. GlMYBs phylogenetic tree.

(A) GlMYB4 phylogenetic tree. (B) GlMYB88 phylogenetic tree.

Fig 4. GlMYBs subcellular localization analysis.

DAPI is used as the nuclear dye, and YFP is the fluorescent protein carried by the target gene. Bright-field image of the cellular morphology under unexcited light. Merged represents the overlapping of all three images. The red arrow indicates the position of the nucleus and GlMYBs protein co-localization. (A) GlMYB4 subcellular localization analysis. (B) GlMYB88 subcellular localization analysis.

Fig 5. Expression analysis of GlMYB4 in different structures and growth periods of licorice.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for aseptic licorice seedling at different structures and growth periods: *P < 0.05; **P < 0.01. (A) Expression analysis of GlMYB4 in different growth periods of licorice. (B) Expression analysis of GlMYB4 in different structures of licorice.

Fig 6. Expression analysis of GlMYB88 in different structures and growth periods of licorice.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for aseptic licorice seedling at different structures and growth periods: *P < 0.05; **P < 0.01. (A) Expression analysis of GlMYB88 in different growth periods of licorice. (B) Expression analysis of GlMYB88 in different structures of licorice.

Fig 7. The expression level of the GlMYB4, GlMYB88, C4S, and CHS genes in the control and overexpression (OE) cells.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for the empty vector control: *P < 0.05; **P < 0.01. Control: untransformed cell; GlMYB4 OE: overexpression GlMYB4 group; GlMYB88 OE: overexpression GlMYB88 group; C4H: cinnamic acid-4-hydroxylase; CHS: chalcone synthase.

Fig 8. Determination of the total flavonoids in the control and experimental groups.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for the empty vector control: *P < 0.05; **P < 0.01. Control: untransformed cell; GlMYB4 OE: overexpression GlMYB4 group; GlMYB88 OE: overexpression GlMYB88 group.

Fig 9. Determination of the isoliquiritigenin in the control and experimental groups.

Data presented here are the mean of three replicates with error bars indicating ± SD. Asterisks indicate significant differences with values for the empty vector control: *P < 0.05; **P < 0.01. Control: untransformed cell; GlMYB4 OE: overexpression GlMYB4 group; GlMYB88 OE: overexpression GlMYB88 group.