A Myb transcription factor, PgMyb308-like, enhances the level of shikimate, aromatic amino acids, and lignins, but represses the synthesis of flavonoids and hydrolyzable tannins, in pomegranate (Punica granatum L.)

Abstract

Pomegranate fruit peels are highly abundant in metabolites derived from the shikimate pathway, such as hydrolyzable tannins (HTs) and flavonoids. These metabolites are beneficial to human health (commercial juice is enriched with peel metabolites), and also protect the fruit from environmental stresses. To understand the transcriptional control of shikimate pathway-related metabolites in pomegranate, we cloned and characterized a subgroup S4 R2R3 Myb transcription factor, PgMyb308-like. Overexpressing PgMyb308-like in pomegranate hairy roots increased the accumulation of shikimate, aromatic amino acids, isoferulic acid, and total lignins, but led to reduced gallic acid and its downstream products HTs, as well as multiple flavonoids. Changes in these metabolites are supported by the increased expression of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and shikimate dehydrogenase 1 (PgSDH1) (the SDH isoform associated with shikimate biosynthesis), and the reduced expression of PgSDH4 (the SDH isoform suggested to produce gallic acid). Transcriptome analysis of PgMyb308-like-overexpressing hairy roots further revealed reprogramming of cell wall-related genes, while overexpression of PgMyb308-like in Arabidopsis thaliana plants uncovered its distinct role in a different genetic and metabolic background. These results together suggest that PgMyb308-like activates genes in the shikimate pathway and lignin biosynthesis, but suppresses those involved in the production of HTs and flavonoids.

Figure 1

The shikimate pathway delivers precursors for aromatic amino acid (AAA), flavonoid, lignin, and hydrolyzable tannin (HT) biosyntheses. Dashed arrows represent multiple biosynthetic steps. Selected enzymes in the shikimate, lignin, and HT pathways are shown. SDH, shikimate dehydrogenase; DAHPS, 3-deoxy-D-arabino heptulosonate-7-phosphate synthase; UGT, UDP-dependent glycosyltransferase; COMT, caffeic acid 3-O-methyltransferase.

Figure 2

Sequence analysis of PgMyb308-like. (A) The amino acid sequence with the conserved motifs. The R2 and R3 domains are indicated by violet and green color. The bHLH-binding domain (within the R3 domain) and the DNEI domain are indicated in gray and orange. The C1 and C2 motifs are indicated in red and blue. The last five amino acids (marked in brown) at the C-terminus differ from the sequence previously deposited in the NCBI (accession number: AIO09658.1). (B) A cladogram of PgMyb308-like and additional R2R3-Myb transcription factors that have similar motives from other plants. The accession numbers are listed in Supplementary Fig. S2.

Figure 3

Expression analysis of transgenic pomegranate hairy roots. The gene expression data were normalized with the pomegranate Ribosomal Protein S gene. Pg1-Pg3, hairy root lines overexpressing PgMyb308-like. EV1-EV3, empty vector control lines. Each hairy root line was derived from an independent transformation event. The data are means of three biological replicates with error bars indicating SD. Different letters indicate significantly different (P < 0.01) expression, based on the Tukey–Kramer test.

Figure 4

The contents of metabolites related to the pathway of shikimate, phenylpropanoid, and hydrolyzable tannin in transgenic hairy roots overexpressing PgMyb308-like (T) or an empty vector (EV). Metabolites related to the synthesis of shikimate and aromatic amino acids are in a gray frame; hydrolyzable tannins are in a blue frame; flavonoids are in a green frame; and hydroxycinnamic acids in a red frame. The Y-axis represents the normalized peak area. The results are average ± SE of three biological repeats of transgenic lines and each biological repeat has three technical repeats. Statistical significance is based on Student’s t-test (P < 0.01, and P < 0.001) and marked by one and two asterisks, respectively. 1,2,6-TGG, 1,2,6-Trigalloylglucose; 1,2,3,6-TGG, Tetra-O-galloyl-β-D-glucose; 2,4-digalloyl-1,3,5-GG, 2,4-O-digalloyl-1,3,6-tri-O-beta-D-galloylglucose; 4-HPP, 4-Hydroxyphenylpyruvate.

Figure 5

Quantitative real-time PCR analysis of genes related to the shikimate pathway. Expression of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (PgDAHPS) as well as shikimate dehydrogenase 1 (PgSDH1), PgSDH3.2, and PgSDH4 is shown. The expression levels were normalized with pomegranate Ribosomal Protein S. The results are average ± SE of three biological replicates of transgenic lines, each with three technical replicates. Statistical significance was determined based on Student’s t-test (P < 0.05 and P < 0.01) and marked with one and two asterisks, respectively.

Figure 6

The data obtained from Arabidopsis thaliana lines overexpressing PgMyb308-like. (A) Relative expression of PgMyb308-like in transgenic lines overexpressing PgMyb308-like (At1-At3) and control (EV) plants. The expression levels were normalized with the actin gene. Each plant line is derived from an independent transformation event. The results are the average of all three transgenic plants, each plant tested in three technical repetitions, with error bars indicating SD. Significantly different (P < 0.05) expression, based on the Tukey–Kramer test, was marked by different letters; (B) Levels of gallic acid, shikimate, chorismate and aromatic amino acids (AAAs) in the transgenic lines as determined by GC–MS analysis; (C) Expression analysis of AtSDH and AtDAHPS in the transgenic lines. Statistical significance was determined based on Student’s t-test (P < 0.05 and P < 0.01) and marked with one and two asterisks, respectively.