The Scutellaria baicalensis R2R3-MYB Transcription Factors Modulates Flavonoid Biosynthesis by Regulating GA Metabolism in Transgenic Tobacco Plants

R2R3-MYB proteins play role in plant development, response to biotic and abiotic stress, and regulation of primary and secondary metabolism. Little is known about the R2R3-MYB proteins in Scutellaria baicalensis which is an important Chinese medical plant. In this paper, nineteen putative SbMYB genes were identified from a S. baicalensis cDNA library, and eleven R2R3-MYBs were clustered into 5 subgroups according to phylogenetic reconstruction. In the S. baicalensis leaves which were sprayed with GA3, SbMYB2 and SbMYB7 had similar expression pattern with SbPALs, indicating that SbMYB2 and SbMYB7 might be involved in the flavonoid metabolism. Transactivation assay results showed that SbMYB2 and SbMYB7 can function as transcriptional activator. The expression of several flavonoid biosynthesis-related genes were induced or suppressed by overexpression of SbMYB2 or SbMYB7 in transgenic tobacco plants. Consistent with the change of the expression of NtDH29 and NtCHI, the contents of dicaffeoylspermidine and quercetin-3,7-O-diglucoside in SbMYB2-overexpressing or SbMYB7-overexpressing transgenic tobacco plants were decreased. The transcriptional level of NtUFGT in transgenic tobacco overexpressing SbMYB7 and the transcriptional level of NtHCT in SbMYB2-overexpressing tobacco plants were increased; however the application of GA3 inhibited the transcriptional level of these two genes. These results suggest that SbMYB2 and SbMYB7 might regulate the flavonoid biosynthesis through GA metabolism.


Introduction
MYB proteins present in all eukaryotes and play roles in a variety of plant-specific processes, as evidenced by their extensive functional characterization in Arabidopsis (Arabidopsis thaliana) [1], maize (Zea mays) [2], rice (Oryza sativa) [3], petunia (Petunia hybrida) [4], grapevine (Vitis vinifera L.) [5], poplar (Populus tremuloides) [6] and apple (Malus domestica) [7]. The increasing availability of plant genome sequence information has allowed comparisons and a better understanding of the evolution of this large family of transcription factors.
Most plant MYB proteins belong to the R2R3-MYB subfamily [8], and the Arabidopsis R2R3-type MYB factors encoded by the AtMYB genes have been categorized into 22 subgroups on the basis of the conserved amino acid sequence motifs [9].
Arabidopsis R2R3-MYB proteins have been found to be involved in primary and secondary metabolism, cell fate and identity, developmental processes and responses to biotic and abiotic stresses [10]. Some R2R3-MYB proteins are also involved in the regulation of the flavonoid biosynthetic pathway [11]. Overexpression of AtMYB75/PAP1 and AtMYB90/PAP2 resulted in a massive activation of phenylpropanoid biosynthetic genes and enhanced the accumulation of lignin, hydroxycinnamic acid esters, and purple anthocyanins [12]. AtMYB4 was shown to negatively regulate the expression of cinnamate 4-hydroxylase gene, then repress the synthesis of sinapoyl malate.
In our previous work, proteomics analysis showed that the protein level of a putative R2R3-MYB transcription factor in S. baicalensis roots was increased under water deficit condition. This R2R3-MYB has high identity with AtMYB113 which is involved in the regulation of anthocyanin biosynthesis, indicating that the R2R3-MYB transcription factor is also involved in flavonoid biosynthesis in S. baicalensis. The expression levels of several proteins related to GA metabolism were also affected. These prompted us to consider the possible linkage between flavonoid accumulation and hormone metabolism [23].
In this paper, we obtained nineteen S. baicalensis MYB transcription factors from a cDNA library, and performed a phylogeny and expression patterns analysis to yield an overview of the R2R3-MYB gene family in S. baicalensis. We further confirmed that SbMYB2 and SbMYB7 play roles in flavonoids biosynthesis.

Identification of R2R3-MYB genes from S. baicalensis
We have developed a S. baicalensis full-length cDNA library (unpublished work). To identify R2R3 type MYB genes in S. baicalensis, a preliminary BLASTX search was performed using NR database in full-length cDNA library. Only hits with E values below e -30 were considered as members of this gene family. Eleven SbMYB genes have R2R3-MYB conserved domains and motifs, and their deduced proteins showed different lengths, isoelectric points, and molecular weights (Table S1; Table S2). The sequences of these nineteen SbMYB genes have been submitted to the GenBank with the accession number KC990835, KC990836, KF008651-KF008667.
Based on sequence similarity, the identified S. baicalensis R2R3-MYB proteins were clustered into 5 subgroups, according to clades with at least 50% bootstrap support ( Figure  2). During the subfamily classification of the MYB genes, we also took into account the results of Stracke et al. [8] and Dubos et al. [26] for AtMYBs. The validity of our phylogenetic reconstruction is confirmed by the fact that it shows the same subgroups as those observed in previously constructed phylogenetic trees. SbMYB2, SbMYB7 and SbMYB11 belong to subgroup S14. SbMYB13 and SbMYB19 were clustered with OsMYB4 and ATMYB5, and SbMYB15 was clustered with AtMYB20, AtMYB43, AtMYB85, AtMYB42, AtMYB40 and AtMYB99. Only SbMYB8 belongs to subgroup S6, and SbMYB16 belongs to subgroup S18. In general, the gene functions of a clade appear highly but not absolutely conserved across plant species. Thus, knowledge of the gene functions of certain members will facilitate confirmation of paralogous and orthologous relationships.

The expression pattern of S. baicalensis R2R3-MYB genes and flavonoid biosynthesis-related genes
The flavonoid accumulation in S. baicalensis might be related with GA hormone metabolism and some R2R3-MYB proteins might be involved in the flavonoid accumulation [23]. The expression of some R2R3-MYB genes and the flavonoid biosynthesis-related genes were investigated in the S. baicalensis leaves which were sprayed with GA 3 . The results showed that exogenous GA 3 decreased the expression of Sb4CL, SbUBGAT, SbPAL1, SbPAL2 and SbPAL3, whereas the expression of SbC4H and SbCHS were increased by GA 3 treatment ( Figure 3). The expression of SbMYB2, SbMYB5, SbMYB7 and SbMYB12 was decreased after GA 3 treatment, however GA 3 treatment increased the expression of SbMYB8 ( Figure 4). SbMYB2 and SbMYB7 had similar expression pattern with SbPALs, indicating that SbMYB2 and SbMYB7 might be involved in the flavonoid metabolism. The functions of these two genes were further investigated.

Subcellular localization of SbMYB2 and SbMYB7
Firstly, we determine the subcellular localization of SbMYB2 and SbMYB7. The full-length cDNA sequence of SbMYB2 and SbMYB7 were fused in front of the 5' terminus of GFP reporter gene under the control of CaMV 35S promoter with the correct reading frame, respectively. The recombinant constructs of the SbMYB2-GFP and SbMYB7-GFP fusion gene and GFP alone were transformed into onion (Allium cepa) epidermal cells by particle bombardment. SbMYB7-GFP fusion protein accumulated mainly in the nucleus, suggesting that SbMYB7 is a nucleus-localized protein. Whereas SbMYB2-GFP fusion protein is located not only in nucleus but also in some other plastids，and GFP alone was present throughout the whole cell ( Figure 5). These results are consistent with the predicted localization results (Table S3).

Transactivation assay of SbMYB2 and SbMYB7
A yeast GAL4 system was used to determine the transcription activity of SbMYB2 and SbMYB7. The full-length cDNA of SbMYB2 and SbMYB7 was fused to the GAL4 DNA-  binding domain of the pGBKT7 vector, and the fusion plasmid pBD-SbMYB2 and pBD-SbMYB7 was transformed into the yeast strain YGR2. Figure 6 showed that yeasts transformed with pBD-SbMYB2 or pBD-SbMYB7 could grow on the selection synthetic dextrose mediums lacking tryptophan and adenine (SD/-Trp/-Ade), and on the medium lacking tryptophan, adenine, and histidine (SD/-Trp/-Ade/-His). A healthy growth of yeast on both media were detected in the transformants containing the full-length cDNA of SbMYB2 and SbMYB7 compared with the control yeast transformed with empty vector, These results suggests that the SbMYB2 and SbMYB7 protein can function as transcriptional activator.

Molecular characterization of transgenic tobacco lines overexpressing SbMYB2 and SbMYB7
To investigate the function of SbMYBs in plants, SbMYB2 and SbMYB7 were transformed into tobacco plants, respectively. The integration of SbMYB2 and SbMYB7 was confirmed using PCR analysis ( Figure S1). The real-time RT-PCR analysis results showed that the expression of SbMYB2 and SbMYB7 was markedly increased in the transgenic plants  (Table S4). Three independent transgenic lines (e10-25, e10-26 and e10-29) overexpressing SbMYB2 and three independent transgenic lines (e18-53, e18-b and e18-d) overexpressing SbMYB7 were selected for further analysis.

SbMYB2 and SbMYB7 regulates the expression of flavonoid biosynthesis-related genes
To investigate whether SbMYB2 and SbMYB7 regulates the flavonoid biosynthesis, the expression of several flavonoid biosynthesis-related genes including NtPAL1, NtPAL2, NtC4H, , indicating that SbMYB2 negatively regulate the expression of CHI and GT4. The expression of NtAT1 was increased and the transcriptional level of NtDH29 was slightly decreased in both SbMYB2-overexpressing and SbMYB7-overexpressing transgenic tobacco plants ( Figure 8). Because NtDH29 is an important gene which is involved in the biosynthesis of dicaffeoylspermidine [20], above results indicates that SbMYB2 and SbMYB7 could be related to the dicaffeoylspermidine formation. The expression of flavonoid related genes in wild type tobacco plants were not affected by GA treatment (Table  S5).

Exogenous GA 3 affected the expression of flavonoid pathway genes in transgenic tobacco plants overexpressing SbMYB2 or SbMYB7
To further analyze the function of SbMYB2 and SbMYB7 in possible linkage among flavonoid accumulation and GA metabolism, exogenous GA 3 were sprayed on the leaves of transgenic plants overexpressing SbMYB2 or SbMYB7, and the expression of several flavonoid pathway genes were measured by real-time RT-PCR. Expression of NtPAL1 was decreased in both SbMYB2-overexpressing and SbMYB7overexpressing transgenic plants at 2 and 3h after spay exogenous GA 3 . The expression of NtPAL2 was markedly increased in SbMYB2-overexpressing transgenic plants at 1h after spaying exogenous GA 3 , whereas was increased in SbMYB7-overexpressing transgenic plants at 2h after exogenous GA 3 treatment. Transcriptional level of NtCHS was increased in SbMYB2 and SbMYB7-overexpressing transgenic plants at 3h after spaying exogenous GA 3 . The transcriptional levels of NtCHI and NtUFGT were decreased at 1h and increased at 2 and 3h in SbMYB2-overexpressing transgenic plants after spaying exogenous GA 3 , and decreased at 1h in SbMYB7-overexpressing transgenic plants after spaying exogenous GA 3 . The expression of NtHCT was decreased in SbMYB2-overexpressing transgenic plants at 3h after spaying exogenous GA 3 (Figure 8). Gene expression pattern without GA application was also investigated and no difference was observed (Table S6).

SbMYBs bind with the box L sequence of the PAL promoter
Several reports have shown that MYB proteins regulate the expression of PAL by combining the box L [24], and box L is present in the NtPAL gene [25]. The box L sequence in the promoter sequence of NtPAL (GenBank:AB008199) was predicted as ACTTTG using Softberry (linux1.softberry.com). The sequence contains ACTTTG, which has been identified in several gene promoters as a component of binding sites for transcription factor. The interaction between SbMYB2 and SbMYB7 with NtPAL promoter sequence was assayed with electrophoretic mobility shift assay (EMSA) experiment. SbMYB2 and SbMYB7 were expressed in E. coli, respectively, for the use of EMSA analysis. No binding bands were detected with crude proteins of E. coli without or with empty vector (Figure 10 lane 1 and lane 2). SbMYB2 and SbMBY7 specifically bind with the box L sequence, and unlabeled probes inhibit the binding (Figure 10). These results confirmed that SbMYB proteins could combine to the box L sequence of NtPAL gene which is the target gene of MYB protein.
In this study, we isolated nineteen full-length SbMYB genes from a S. baicalensis cDNA library, and eleven R2R3-MYBs with conserved R2R3 domain were divided into 5 subgroups based on the conservation of the DNA binding domain of Arabidopsis MYB proteins. Analysis of new plant genomes suggests that some MYB genes have evolved to fulfill lineagespecific functions [29]. Despite the divergence of the aminoacid sequence outside of the MYB domain, there are some conserved motifs that may contribute to function. Within subgroups that are conserved between divergent species, primary protein structures and biological functions are correlated, such as phenylpropanoid metabolism regulation by R2R3-MYB subgroups 6 [9]. Therefore, protein structure and gene expression patterns can help deduce the functions of new MYB proteins in plants. Using this way, some Arabidopsis MYB proteins were predicted to have the function of controlling flavonoids [30] and flavonol [31] biosynthesis. In this study, the transcriptional levels of SbMYB2 and SbMYB7 which belong to subgroup 14 were decreased by GA 3 treatment, whereas the expression of SbMYB8 which belongs to subgroups 6 was increased after spraying exogenous GA. The transcriptional levels of SbCHS and SbC4H, two key genes which are involved in baicalein biosynthesis, were also increased. SbCHS and SbC4H have the similar expression pattern with SbMYB8, indicating that SbMYB8 was involved in the flavonoid biosynthesis in S. baicalensis based on subgroup classification and co-expressed analysis.
MYB proteins in subgroup 14 were believed to have functions on plant development [32]. Here, SbMYB2 and SbMYB7 which belong to subgroup 14 co-expressed with SbPALs, indicating that SbMYB2 and SbMYB7 might be involved in the flavonoid metabolism. To confirm this hypothesis, transgenic tobacco plants overexpressing SbMYB2 or SbMYB7 were developed. In transgenic plants, the transcriptional level of some flavonoid biosynthesis-related genes (NtPAL1, NtPAL2, NtC4H and NtUFGT) were increased, whereas the transcription levels of NtCHI and NtGT4 were decreased, suggesting that SbMYB2 and SbMYB7 could upregulate the first step and down-regulate the last step of flavonoid biosynthesis. Consistent with the decreased expression of NtDH29 and NtCHI, the content of dicaffeoylspermidine and quercetin-3,7-O-diglucoside in transgenic tobacco plants was significantly decreased by overexpression of SbMYB2 or SbMYB7, suggesting phenylpropanoid-polyamine conjugates was negatively regulated by above SbMYBs. Dicaffeoylspermidine was a phenylpropanoid-polyamine conjugates and it has been shown that the N-coupling reaction of polyamines to phenolic acids (such as cinnamic, p-coumaric, caffeic, ferulic and sinapic acids) in plants is catalyzed by a specific class of acyltransferase enzymes. NaMYB8 silencing induces specific alterations in the accumulation of coumaroyl-containing metabolites and suppresses caffeoyl-and feruloyl-containing metabolites, and resulted in a strong suppression of dicaffeoylspermidine in Nicotiana [33].
Plant hormones affect the accumulation of secondary metabolites and R2R3-type MYB proteins also participate in mediating hormone actions [34]. It has been observed that ABA and GA 3 treatment decreased CsPAL expression level and catechin content [35]. GA 3 may inhibit the phenylpropanoid pathway through affecting PAL in Myrica rubra, pea and carrot [36][37][38]. Devaiah et al. [39] reported that AtMYB62 regulated phosphate starvation responses via changes in GA metabolism and signaling. Gibberellin acts through jasmonate to control the expression of AtMYB21, AtMYB24, and AtMYB57 to promote stamen filament growth in Arabidopsis [40]. Rice GaMYB is an important component of GA signaling in cereal aleurone cells and anther development [41]. In our previous study, the levels of total flavonoids and baicalin and the ratio of baicalin to baicalein in roots of S. baicalensis were decreased under water deficit condition after application of GAs, and these decreases were recovered after application of paclobutrazol [23]. The results in this paper also insist that GAs affected flavonoid metabolism in S. baicalensis. Over-expression of Arabidopsis MYR1 or MYR2 produced GA-deficient symptoms that were rescued by application of GA 3 [42]. Our result also showed that the transcription level of NtUFGT in SbMYB7-overexpressing tobacco and NtHCT transcripts in SbMYB2-overexpressing tobacco was increased. These changes could be rescued by application of GA 3 . These results suggest that SbMYB2 and SbMYB7 might regulate the flavonoid biosynthesis through the negative effect on levels of bioactive GA.
ZmC1 regulates anthocyanin production together with ZmR in maize, suggesting that R2R3 MYB proteins are often involved in the combinatorial interaction of transcription factors for the generation of highly specific expression patterns [43]. The transcriptional levels of NtPAL1 and NtPAL2 were increased in transgenic tobacco plants overexpressing SbMYB2 or SbMYB7. NtPAL2 transcription level was also increased in transgenic plants overexpressing SbMYB2 and SbMYB7 after GA 3 treatment. It has been reported that R2R3-MYB factors regulate the transcriptional activation of Pinus pinaster PAL by interaction with the promoter sequence containing AC elements [44], and MYB proteins regulate the expression of PAL by combining with the box L [24]. EMSA analysis clearly showed that SbMYB2 and SbMYB7 could combine to the box L sequence of NtPAL gene which is the target gene of MYB protein (Figure 10).
Within a subgroup, paralogs can control the same metabolic pathway in different cell types as a result of differences in expression patterns [27,30]. AtMYB66/WER and GL1, both clustering together in subgroup 15, can functionally complement each other and display different biological functions only because of their different spatial expression patterns [45]. Although both SbMYB2 and SbMYB7 belong to subgroup 14, they have 62.1% identity at nucleotide acid level and 45.8% identity at amino acid level, and they have different subcellular localization. The transcriptional level of flavonoid biosynthesis-related genes in SbMYB2-and SbMYB7overexpressing transgenic tobacco after spray GA 3 have different expression patterns, indicate that SbMYBs play redundant, but divergent roles in flavonoid biosynthesis and GA response. The results in this study suggested that SbMYB2 and SbMYB7 could affect phenylpropanoid biosynthesis, and SbMYB2 affects flavonoid accumulation through regulating the gibberellin (GA) signaling pathways.

Plant Materials and Growth Condition
The seeds of S. baicalensis were obtained from Institute of Chinese Materia Medica, Academy of Chinese Medical Sciences, Beijing, China), sterilized in 0.5% NaOCl for 5 min, then washed 3 times with sterile water, and placed in petri dishes to germinate. The seedlings 2 weeks after germination were transferred to individual pots (10 seedlings per pot) containing 500 g dried soil in climate chamber at 25°C with 16 h-light photoperiod under well-water condition. GA 3 (100 uM) were sprayed on leaves of plants one week after transplant of S. baicalensis and transgenic tobacco. The leaves were sampled three times at 1, 2 and 3 h after spraying. The sample were rinsed three times in distilled water, and then stored at -80°C for further experiments

Identification of R2R3-MYB Protein in S. baicalensis
To identify R2R3-MYB genes, we performed a BLASTX algorithm [46] at the S. baicalensis full-length cDNA library (Yuan et al, unpublished) against the amino acid sequences in NR database (http://www.ncbi.nlm.nih.gov). The functional and structural domains were predicted by InterProScan [47] and Blast2GO [48] analysis, respectively.

Construction of the Phylogenetic Trees
Phylogenetic analysis of the alignments was performed using ClustalW (Thompson, 1994) and MEGA 4.0 [52] for neighborjoining analysis. The reliability of these tree topologies was evaluated using bootstrap support with 1000 replicates [53]. The sequences of 126 Arabidopsis R2R3-MYB proteins were downloaded from the TAIR Arabidopsis genome (http:// www.arabidopsis.org/). The predicted proteins of 52 wellknown plant R2R3-MYB genes were collected from the National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov/).

Gene expression analysis in S. baicalensis
Total RNA was extracted from plant tissues using Trizol reagent (Invitrogen, USA) and pretreated with RNase-Free DNase (Promega, USA) to eliminate genomic DNA contamination. RNA integrity was analyzed on 1% agarose gel. RNA quantity was determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA).

Subcellular localization
The whole coding sequence of SbMYB2 and SbMYB7 was ligated into pE3025 vector [54] digested with EcoRI and KpnI to generate plasmids pGEM-SbMYB2 and pGEM-SbMYB7 ， respectively. In both plasmids, SbMYB-GFP fusion genes are under the control of CaMV 35S promoter. The construct was confirmed by sequencing and used for transient transformation of onion (Allium cepa) epidermis via a gene gun (Bio-Rad). After 24 h of incubation, GFP fluorescence in transformed onion cells was observed under a confocal microscope (Zeiss, Germany).

Transactivation assay
To determine the transactivation activity, the open reading frames of SbMYB2 and SbMYB7 were generated by PCR amplification, cloned into vector pBD-GAL4 which was digested with EcoRI and SalI, to construct pBD-SbMYB2, and pBD-SbMYB7, respectively. The constructs were transformed into YGR2 cells by the lithium acetate-mediated method. The transformed yeast strains were placed on SD/-Trp medium at 28 °C for 2 days. Yeast transformants from SD medium lacking Trp were then transferred and streaked onto solid SD agar lacking Trp/His/Ade (SD/-Trp/-His/-Ade) to score the growth response after 3 days. For the colony-lift filter assay (X-gal assay), the yeast was transferred to Whatman filter paper plus X-gal for transcription activation activity analysis within 8 h. pGAL4 and pBD-GAL4 was used as a positive control and negative control, respectively.

Tobacco transformation
SbMYB2 and SbMYB7 fragments were inserted into binary vectors, pCambia1305 to produce p35Spro-SbMYB2 and p35Spro-SbMYB7, respectively. The constructs were then transformed into Agrobacterium tumefaciens EHA105. Tobacco (Nicotiana tabacum) leaf discs were transformed via an A. tumefaciens mediated leaf disc procedure [55] and selected using 50 mg L -1 Hygromycin B and 200 mg L -1 carbenicillin. After rooting and acclimatization, regenerated plants were grown in a greenhouse to set seeds by self-pollination. T1 transgenic plants were used for further analyses.

Chemical analysis
To determine flavonoid content, 100 mg powdered tobacco leaf was extracted for 1 h in 1 mL ethyl alcohol. The solution was filtered through a membrane filter (0.2 μm), and flavonoid concentrations were determined using an UPLC-Q-Tof system with a 1.0 mL/min flow rate. UPLC was performed on a diamonsil C18 column (4.6 mm×250 mm, 5 μm). The detection wavelength was set at 354 nm and the column temperature was maintained at 30°C. The mobile phase consisted of acetonitrile-methanoic acid (A; 99.9:0.1, v/v) and deionized water-trifluoroacetic acid (B; 99.9:0.1, v/v). The initial condition

Expression of SbMYBs protein in E.coli
The open reading frame (ORF) of SbMYB2 and SbMYB7 was cloned into the expression vector pGEX-4T-1 and transformed into Transetta (DE3) chemically competent cells (Beijing TransGen Biotech Co., Ltd, China), respectively. The vector pGEX-4T-1 (+) allows in-frame cloning of PCR products resulting in a GST-tag attached at the N-terminal end of the recombinant protein. Expression of the recombinant protein was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) and cells were harvested at 9h.

Electrophoretic Mobility Shift Assay
The boxes L sequence in the promoter sequence of NtPAL (GenBank:AB008199) was as ACTTTG using Softberry (linux1.softberry.com). Oligonucleotides of boxes L sequence were synthesized and labeled with biotin (Sangon Biotech (Shanghai) Co., Ltd., China) for chemiluminescence using a lightshift chemiluminescent electrophoretic mobility shift assay kit (Pierce). After labeling, complementary labeled strands were mixed together in an equimolar ratio and annealed at room temperature after denaturation at 90°C. Gel mobility shift assays were performed by incubating 0.5 ng of labeled probe with SbMYBs protein and competing oligonucleotides in binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, and 1 μg/μl poly(dIdC) at room temperature for 30 min. Mixtures were sizefractionated on a non-denaturing 46% polyacrylamide gel followed by drying and transfer to nitrocellulose membranes and detection by streptavidin-HRP/chemiluminescence for biotin-labeled probes.