SmMYB111 is a key factor to phenolic acid biosynthesis and interacts

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Biotechnology and Biological Transformations

SmMYB111 is a key factor to phenolic acid biosynthesis and interacts with both SmTTG1 and SmbHLH51 in Salvia miltiorrhiza Shasha Li, Yucui Wu, Jing Kuang, Huaiqin Wang, Tangzhi Du, Yaya Huang, Yuan Zhang, Xiaoyan Cao, and Zhezhi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02548 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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Journal of Agricultural and Food Chemistry

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SmMYB111 is a key factor to phenolic acid biosynthesis and interacts

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with both SmTTG1 and SmbHLH51 in Salvia miltiorrhiza

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Shasha Li1#, Yucui Wu2#, Jing Kuang3, Huaiqin Wang1, Tangzhi Du1, Yaya Huang1, Yuan

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Zhang1, Xiaoyan Cao1*, Zhezhi Wang1*

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1

6

in Northwest of China, Key Laboratory of the Ministry of Education for Medicinal

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Resources and Natural Pharmaceutical Chemistry, Shaanxi Normal University, Xi’an

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710062, China;

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2

National Engineering Laboratory for Resource Development of Endangered Crude Drugs

School of Landscape and Ecological Engineering, Hebei University of Engineering,

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Handan 056038, China;

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3

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# These authors contributed equally to this work.

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*Authors for correspondence:

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Xiaoyan Cao (Tel: 86-29-85310266 Email: [email protected] ORCID ID:

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https://orcid.org/0000-0002-3188-6663)

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Zhezhi Wang (Tel: 86-29-85310260 Email: [email protected]

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https://orcid.org/0000-0002-6111-1551)

Ningxia Polytechnic, Yinchuan 750001, China;

18

1

ACS Paragon Plus Environment

ORCID ID:


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Abstract

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Transcription factors that include myeloblastosis (MYB), basic helix–loop–helix (bHLH),

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and

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the phenylpropanoid pathway. However only a few MYB and bHLH members involved in

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the biosynthesis of salvianolic acid B (Sal B) have been reported, and little is known about

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Sal B pathway regulation by the WD40 protein transparent testa glabra1 (TTG1)-dependent

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transcriptional complexes in Salvia miltiorrhiza. We isolated SmTTG1 from that species for

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detailed functional characterization. Enhanced or reduced expression of SmTTG1 was

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achieved by gain- or loss-of-function assays, respectively, revealing that SmTTG1 is

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necessary for Sal B biosynthesis. Interaction partners of SmTTG1 protein were screened by

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yeast two-hybrid (Y2H) assays with the cDNA library of S. miltiorrhiza. A new

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R2R3-MYB transcription factor, SmMYB111, was found through this screening.

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Transgenic plants over-expressing or showing reduced expression of SmMYB111

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up-regulated or deregulated, respectively, the yields of Sal B. Both Y2H and Bimolecular

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Fluorescent Complementation experiments demonstrated that SmMYB111 interacts with

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SmTTG1 and SmbHLH51, a positive regulator of the phenolic acid pathway. Our data

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verified the function of SmTTG1 and SmMYB111 in regulating phenolic acid biosynthesis

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in S. miltiorrhiza. Furthermore, ours is the first report of the potential ternary transcription

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complex SmTTG1–SmMYB111–SmbHLH51, which is involved in the production of Sal B

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in that species.

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Keywords: Salvia miltiorrhiza; salvianolic acid B; SmTTG1; SmMYB111; transcription

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complex.

WD-repeat

protein,

which

often

forms

a

41 2


ternary

complex

to

regulate

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Introduction

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Salvia miltiorrhiza Bunge is a perennial medicinal plant with great medicinal and

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economic value.1 Its dry roots have been used for more than two thousand years in

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traditional Chinese medicine (TCM) to treat various diseases, such as cardiovascular

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diseases, coronary heart diseases, dysmenorrhea, and amenorrhoea.2, 3 It presents a model

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plant system for TCM research because of its high transformation efficiency, short life

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cycle, published whole genome data, and reliable therapeutic actions.4 The active

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pharmaceutical ingredients of danshen include two major groups: lipophilic tanshinones5

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and hydrophilic salvianolic acids.6 Among the phenolic acids, salvianolic acid B (Sal B)

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is predominant and is regarded as a powerful natural product

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properties and scavenging of free radicals.7 It offers protection against fibrosis, tumor

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development, aging, and cardiovascular/cerebrovascular diseases.7, 8

for its antioxidant

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The biosynthetic pathway of Sal B is thought to include both phenylpropanoid and

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tyrosine-derived pathways.9 The former is a general pathway for the synthesis of phenolic

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acids, flavonoids (e.g., anthocyanins and flavones), and lignins.9, 10 Although Sal B is an

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important medicinal component, only relatively low levels are produced by S. miltiorrhiza

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plants. Therefore, biological approaches have been taken to augment its synthesis,

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including the engineering of genes in the biosynthetic pathway and ectopic expression of

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transcription factors (TFs).11-15 For example, ectopic overexpression of AtPAP1, a MYB

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TF gene from Arabidopsis thaliana, can strongly enrich Sal B in S. miltiorrhiza.11

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Myeloblastosis (MYB) TF plays a key role in regulating plant flavonoid biosynthesis

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that interact with basic helix–loop–helix (bHLH) and transparent testa glabra1 (TTG1), a 3



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WD40 repeat protein, to form MBW complexes and control the phenylpropanoid

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pathway.16-21 Arabidopsis bHLHs from subgroup IIIf (including GL3, EGL3, MYC1, and

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TT8) and MYBs (e.g., PAP1/2/3/4, TT2, AtMYB5, and AtMYB4) directly interact with

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TTG1 to form MBW complexes and play a transcriptional role in the biosynthesis of

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flavonoids.17, 22, 23 Wan et al.24 have shown that PtrMYB57–bHLH131–PtrTTG1 negatively

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regulates the biosynthesis of anthocyanins and proanthocyanidins in Populus trichocarpa.

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Although a few endogenous MYBs and bHLHs are reported to be involved in modulating

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Sal B biosynthesis,25-30 little is known about this regulatory role by TTG1 and

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TTG1-dependent transcriptional complexes in S. miltiorrhiza.

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The WD40 repeat protein TTG1 is encoded by single-copy genes, which is the common

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denominator of overlapping regulatory complexes.31,

32

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controlling development and secondary metabolism, such as the formation of trichomes,

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synthesis of flavonoids, seed coat mucilage, and root hair development.32 This TTG1

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commonly acts as a scaffold for the combinational interactions between the MYB and

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bHLH, which are indispensable for regulating the transcriptional activity of the MBW

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complex.33, 34

It only exists in higher plants,

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Here, we explored the function of SmTTG1 in S. miltiorrhiza through experiments with

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overexpression and RNAi-mediated silencing. Our results indicated that SmTTG1 is

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necessary for Sal B biosynthesis. Using SmTTG1 as bait, we then screened a new MYB TF

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from the S. miltiorrhiza cDNA library by yeast two-hybrid (Y2H) assays and named it

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SmMYB111. Transgenic plants over-expressing or silencing SmMYB111 significantly

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induced or reduced, respectively, the accumulation of Sal B. Both Y2H and Bimolecular 4


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Fluorescent Complementation (BiFC) experiments demonstrated that SmMYB111 interacts

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not only with SmTTG1 but also with SmbHLH51, a positive regulator of this phenolic acid

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pathway. We speculated that SmMYB111 positively regulates the synthesis of phenolic

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acids -- Sal B and rosmarinic acid (RA) -- in S. miltiorrhiza by forming SmTTG1–

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SmMYB111–SmbHLH51 ternary transcription complex.

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Materials and Methods

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Culture and Treatment of S. miltiorrhiza

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Seeds of S. miltiorrhiza were collected from the experimental fields of Shaanxi Normal

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University. Sterile plantlets were produced as previously described35. Roots, stems, leaves,

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and flowers from two-year-old greenhouse-grown plants were collected in July for RNA

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extraction. Standards of RA and Sal B were prepared as we described previously.11 All the

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primers used in the study are listed in Table S1.

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Two-month-old S. miltiorrhiza seedlings were sprayed with 500 µM methyl jasmonate

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(MeJA), 500 µM salicylic acid (SA), or 50 µM gibberellin (GA) (100 mL per plantlet).

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Roots were sampled after 0, 2, 6, 8, 12, 48, and 72 h of treatment and each sample included

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six plantlets. Plantlets receiving only sterile water were used as controls.

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Isolation and Sequence Analysis of SmTTG1 and SmMYB111

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Genomic DNA and transcriptome cDNA was obtained as we described previously.36

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SmTTG1 (GenBank Accession Number JX416285) and SmMYB111 (GenBank Accession

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Number ASV64719.1) were cloned from the S. miltiorrhiza genomic DNA and

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transcriptome cDNA with primers SmTTG1-F/R and SmMYB111-F/R, respectively. The

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amplified products were inserted into the pMD19-T simple vector (TaKaRa) and 5



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followed by sequencing.

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Homologous protein sequences of SmMYB111 were aligned by CLUSTAL W (version

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1.83). We constructed the phylogenetic tree by Molecular Evolutionary Genetics

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Analysis software (MEGA 5.2) using the Neighbor-Joining method. The GenBank

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accession numbers for all genes are listed in Table S2.

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Yeast Two-hybrid Screening

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The open reading frame (ORF) of SmTTG1 was amplified with primers 207‒

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SmTTG1-F/R and cloned into donor vector pDONR207 to construct entry vector

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pENTR207‒SmTTG1, and then transferred into pGBKT7 (BD) to construct pGBKT7‒

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SmTTG1 according to the Gateway manufacturer’s protocol (Invitrogen, USA).

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The autoactivation and toxicity test of bait protein SmTTG1 in yeast was performed as

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described by Pan et al.37 Briefly, we transformed bait plasmid pGBKT7‒SmTTG1 into

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AH109 yeast cells according to the instructions in the HighTM AH109 Competent Cell

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Manual (Clontech, USA). Transformants were then put on agar plates containing

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appropriate selection media, i.e., SD/-Trp/X-α-gal or SD/-Trp/-His/-Ade/X-α-gal, then

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incubated at 29°C for 3 d before recording the growth. The pGBKT7 was used as a control.

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To screen the host proteins that interact with SmTTG1, we used Y187 yeast cells

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expressing the S. miltiorrhiza cDNA library to mate with AH109 cells expressing SmTTG1,

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following the protocols described previously.37

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Yeast Two-hybrid Assays

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The ORFs of SmTTG1, SmMYB111, and SmbHLH51 (GenBank Accession Number

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KT215166) were cloned into the donor vector pDONR207 to generate entry vector 6


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pENTR207‒SmTTG1, pENTR207‒SmMYB111, and pENTR207‒SmbHLH51, respectively.

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They were then transferred into destination vector pGADT7 or pGBKT7 to construct

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pGADT7‒SmTTG1/SmMYB111 or pGBKT7‒SmTTG1/SmbHLH51/SmMYB111 according

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to the Gateway protocol. The resulting pGADT7 and pGBKT7 fusion constructs were

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co-transformed into yeast strain AH109 as described by Gietz and Schiestl.38 The

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interaction assays were performed based on the manufacturer’s protocol of Matchmaker

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Gold Yeast Two-Hybrid System (Clontech), and Y2H images were taken on Day 5 of

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incubation.

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Bimolecular Fluorescent Complementation and Subcellular Localization

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Primers 207‒SmTTG1-F/Rʹ, 207‒SmMYB111-F/Rʹ, and 207‒SmbHLH51-F/Rʹ were

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designed (stop codons removed) to clone SmTTG1, SmMYB111, and SmbHLH51 into the

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donor

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pENTR207‒SmMYB111ʹ, and pENTR207‒SmbHLH51ʹ, respectively.

vector

pDONR207

and

construct entry

vectors

pENTR207‒SmTTG1ʹ,

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For BiFC assays, pENTR207‒SmTTG1ʹ, pENTR207‒SmMYB111ʹ, and pENTR207‒

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SmbHLH51ʹ were recombined into pEarleyGate202-YC (YC) or pEarleyGate201-YN (YN)

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to construct BIFC expression vectors YC‒SmTTG1, YN‒SmMYB111, YN‒SmbHLH51 and

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YC‒SmbHLH51. The YC and YN recombinant plasmids were mixed at equal density

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before co-transformation. Co-transformation of YC‒SmTTG1+YN, YN‒SmMYB111+YC,

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and YC‒SmbHLH51+ YN served as negative controls.

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For subcellular localizations, pENTR207‒SmTTG1ʹ and pENTR207‒SmMYB111ʹ

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were recombined into destination vector pEarleyGate10339 to construct subcellular

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localization

expression

vectors

pSmTTG1‒GFP

and

7


pSmMYB111‒GFP.

The


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pEarleyGate103 was used as a positive control.

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Vectors were bombarded into onion epidermal cells using Gene Gun PDS-1000

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(Bio-Rad, USA) at a helium pressure of 1100 psi. After 16 to 24 h of incubation

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at 28°C in a dark chamber, the GFP fluorescence images from living cells were observed

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using a Leica DM6000B microscope (Leica, Germany) with an excitation wavelength

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of 475 nm.

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Construction of Plant Expression Vectors and Plant Transformation

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To obtain SmTTG1 and SmMYB111 overexpression vectors pEarleyGate 202‒SmTTG1 and

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pEarleyGate 202‒SmMYB111, we used pENTR207‒SmTTG1 and pENTR207‒SmMYB111

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to recombine SmTTG1 and SmMYB111 into the destination vector pEarleyGate 202. 39

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SmTTG1-RNAi plasmid was constructed according to the methods we established

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previously.36, Briefly,the 279-bp fragment of SmTTG1 cDNA (coding sequence positions

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44-323 bp) were digested with BamHI/XbaI and KpnI/XhoI (TaKaRa) and inserted into

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vector pKannibal40 to generate pK‒SmTTG1. An interfering box was inserted into pART27

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to generate RNAi vector pSmTTG1–RNAi.

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To construct the amiRNA-mediated SmMYB111-silenced recombinant expression

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vector, we designed a pair of amiRNA oligonucleotide primers, amiR–SmMYB111-F/R,

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using P‒SAMS software (http://p-sams.carringtonlab.org/). This synthesized amiRNA

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cassette was then inserted into the pMDC123SB-AtMIR390a-B/c vector by BsaI

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digestion. 41

172 173

S. miltiorrhiza transformation was performed based on protocols established in our laboratory. 42 8


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Molecular Detection of Transgenic Plantlets

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The CaMV35S promoter was amplified from genomic DNA to check whether the gene had

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been integrated into the transgenic plant genome, using previously published protocols.36

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Total RNA from the roots of S. miltiorrhiza transgenic and wild-type (WT) plants was

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extracted and converted into cDNA. Gene expression was monitored via qRT-PCR on a

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LightCycler® 96 real-time PCR detection system (Roche, Switzerland), with housekeeping

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gene SmUbiquitin serving as an internal reference. Relative expression level of each gene,

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expressed as fold change relative to controls, was calculated by the comparative CT

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method.43

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Based on the transcriptional levels of SmMYB111, we conducted qRT-PCR to determine

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the expression levels of key enzyme genes for the biosynthetic pathway of salvianolic

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acids.

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Determination of Phenolics Concentrations by LC/MS Analysis

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Roots from two-month-old transgenic plants of S. miltiorrhiza were collected and dried at

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20±2°C. The phenolic compounds were extracted as described previously.11

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We determined the content of RA and Sal B on an Agilent 1260 HPLC system coupled

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to an Agilent 6460 QQQ LC-MS system. The extracts were diluted 1000 times and

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separated on a Welch Ultimate XB-C18 column (2.1 × 150 mm, 3 µm). Chromatographic

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separations were performed over a 10-min time span, using organic mobile phase

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acetonitrile as solvent A and aqueous mobile phase with 0.1% formic acid (v/v, in

194

deionized water) as solvent B. The linear gradient comprised 20% to 60% A (0–6 min),

195

60% to 20% A (6–7 min), and 20% to 20% A (7–10 min). The flow rate was set at 9



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0.4 mL/min. Conditions for mass spectrometry were as follows: negative mode; drying

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gas flow rate was set at, 10 L/min and temperature 300°C; nebulizer pressure, 45 psi; ion

198

spray voltage, 3500 V; and sheath gas of 11 L/min, at a temperature of 350°C. Retention

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times were 3.8 min for RA and 4.1 min for Sal B. The precursor/product ion of RA is

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359.1>161; Sal B is 717.2>519.2. We established standard curves of authentic reference

201

standards to quantify these phenolics concentrations.

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Determination of Anthocyanin Concentrations

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Anthocyanin concentrations were determined from the roots of two-month-old transgenic

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and WT S. miltiorrhiza, following quantification protocols that we have described

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previously.11

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Results

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Generation

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miltiorrhiza Plants

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Transgenic plants of S. miltiorrhiza that either over-expressed or suppressed SmTTG1

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were confirmed by PCR, which contain an expected 901-bp fragment of the CaMV35S

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promoter (Figure S1). The expression level of SmTTG1 in the transgenics was examined

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by qRT-PCR (Figure 1). Two independent SmTTG1-overexpressing lines (OT-10 and

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OT-14) with significantly elevated SmTTG1 expression and two SmTTG1-RNAi lines

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(iT-2 and iT-3) with significantly reduced expression were selected for further analysis.

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The controls for this analysis were untransformed WT plants (CK).

of

SmTTG1-overexpressing

and

SmTTG1-RNAi

Transgenic

S.

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Compared with the CK, the transgenic plantlets showed obvious phenotypic changes.

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Whereas stems from CK and OT samples were partially red, those from iTs were 10


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completely green. Anthocyanin concentrations were significantly lower in the roots from

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iT plants than from the CK, which indicated that SmTTGl is involved in the regulation of

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anthocyanin biosynthesis in S. miltiorrhiza. Furthermore, the stems from OT plants had

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significantly more trichomes when compared with the CK, while the iT plants had very

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few trichomes (Figure 1), indicating that SmTTG1 favors trichome formation.

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SmTTG1 Regulates the Production of Sal B and RA in S. miltiorrhiza

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Results from LC/MS showed that the concentrations of RA and its dimer Sal B in the

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roots of two-month-old S. miltiorrhiza plantlets did not differ significantly between CK

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samples and overexpressing lines OT-10 and OT-14 (Figure 2). However, when

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compared with CK, the levels of both components were significantly lower (P

SmMYB111 is a key factor to phenolic acid biosynthesis and interacts

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