SmMYB36, a Novel R2R3-MYB Transcription Factor, Enhances Tanshinone Accumulation and Decreases Phenolic Acid Content in Salvia miltiorrhiza Hairy Roots

Abstract

Phenolic acids and tanshinones are two major bioactive components in Salvia miltiorrhiza Bunge. A novel endogenous R2R3-MYB transcription factor, SmMYB36, was identified in this research. This transcript factor can simultaneously influence the content of two types of components in SmMYB36 overexpression hairy roots. SmMYB36 was mainly localized in the nucleus of onion epidermis and it has transactivation activity. The overexpression of SmMYB36 promoted tanshinone accumulation but inhibited phenolic acid and flavonoid biosynthesis in Salvia miltiorrhiza hairy roots. The altered metabolite content was due to changed metabolic flow which was regulated by transcript expression of metabolic pathway genes. The gene transcription levels of the phenylpropanoid general pathway, tyrosine derived pathway, methylerythritol phosphate pathway and downstream tanshinone biosynthetic pathway changed significantly due to the overexpression of SmMYB36. The wide distribution of MYB binding elements (MBS, MRE, MBSI and MBSII) and electrophoretic mobility shift assay results indicated that SmMYB36 may be an effective tool to regulate metabolic flux shifts.

Figure 1

The model for tanshinone and phenolic acid biosynthesis regulation by SmMYB36.

Figure 2

The phylogenetic tree of R2R3-MYB transcription factors. The phylogenetic tree was constructed by maximum likelihood method of MEGA 6.06 based on the multiple sequence alignment using MUSCLE method.

Figure 3

Subcellular localization of SmMYB36 protein. The pA7-GFP (upper lane) and pA7-GFP-SmMYB36 (bottom lane) plasmids were transiently expressed in onion epidermal cells. Fluorescence was observed using a confocal laser scanning microscope at 24 h after incubation. The pictures show bright field, green fluorescent field, DAPI and overlay of three fields from left to right.

Figure 4

The transactivation analysis of SmMYB36. (a) The yeast AH109 containing pDEST-GBKT7 or pDEST-GBKT7-SmMYB36 construct could survive on SD/-Trp medium regardless of the concentration change of 3AT (from 0 mM to 20 mM). (b) While on SD/-Trp/-His/-Ade medium, only the yeast with pDEST-GBKT7-SmMYB36 plasmid grew normally in the absence of 3AT.

Figure 5

The phenotypes of hairy roots. (a) Hairy roots of S. miltiorrhiza induced by A. rhizogenes strain ATCC15834. Hairy roots were cultured in 6,7-V liquid medium for 18 days before being photographed. (b) The colour differences between different extracts of dry hairy roots. (c) The content of total tanshinones in hairy roots. (d) The content of total phenolics in hairy roots. (e) The content of total flavonoids in hairy roots. The pictures show WT (infected by A. rhizogenes strain ATCC15834), EV (infected by A. rhizogenes strain ATCC15834 containing plasmid pK7WG2R-EV), line 36-1, line 36-2, line 36-3 and line 36-4 (infected by A. rhizogenes strain ATCC15834 containing plasmid pK7WG2R-SmMYB36) hairy roots from left to right. (f) The content of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone II A in transgenic and control hairy roots of S. miltiorrhiza. The x represents the values calculated by standard curves when the peak area is zero (18 days). (g) The content of rosmarinic acid and salvianolic acid B in transgenic and control hairy roots of S. miltiorrhiza. The physiological and HPLC analysis both have three biological repeats of transgenic lines and each biological repeat has three technological repeats. The metabolite contents were shown by their means ± SD.

Figure 6

Relative expression level of phenolic acid biosynthesis pathway genes in transgenic hairy roots. Each line has two biological repeats and each biological repeat has three technological repeats. All values are expressed as means ± SD.

Figure 7

Relative expression level of tanshinone biosynthesis pathway genes in transgenic hairy roots. Each line has two biological repeats and each biological repeat has three technological repeats. All values are expressed as means ± SD.

Figure 8

Analysis of SmMYB36 binding to the predicted promoter probes of secondary metabolism pathway genes in S.miltiorrhiza. Arrows indicates the combination between SmMYB36 and probes.