Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39) from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H) and tyrosine aminotransferase (TAT). These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes

Relative quantitative transcripts analysis of enzyme genes in transgenic lines and controls of <i>S. miltiorrhiza</i>.

<p>The results were analyzed using the comparative Ct method and presented as fold-changes compared with the control sample (untransformed control). The <i>S. miltiorrhiza actin</i> gene was used as an internal control to normalize expression levels. The asterisks indicate statistically significant differences (<i>P</i><<i>0.05</i>) compared to the empty vector control. control: untransformed plant; ox-VC, RNAi-VC: empty vector controls of <i>SmMYB39</i>-overexpressing lines and <i>SmMYB39</i>-RNAi lines; ox-3, ox-17, ox-28: <i>SmMYB39</i>-overexpressing lines; RNAi-11, RNAi-19, RNAi-25: <i>SmMYB39</i>-RNAi lines; <i>PAL</i>: phenylalanine ammonia-lyase; <i>C4H</i>: cinnamic acid 4-hydroxylase; <i>4CL</i>: 4-coumaric acid CoA-ligase; <i>TAT</i>: tyrosine aminotransferase; <i>HPPR</i>: 4-hydroxyphenylpyruvate reductase.</p

Enzyme activities analysis of C4H and TAT in transgenic lines and controls of <i>S. miltiorrhiza</i>.

<p>Data presented here are the mean of three replicates with error bars indicating ± SD. The asterisks indicate statistically significant differences (<i>P</i><<i>0.05</i>) compared to the empty vector control. control: untransformed plant; ox-VC, RNAi-VC: empty vector controls of <i>SmMYB39</i>-overexpressing lines and <i>SmMYB39</i>-RNAi lines; ox-3, ox-17, ox-28: <i>SmMYB39</i>-overexpressing lines; RNAi-11, RNAi-19, RNAi-25: <i>SmMYB39</i>-RNAi lines; C4H: cinnamic acid 4-hydroxylase; TAT: tyrosine aminotransferase.</p

Primers used for the quantitative real-time PCR analysis.

<p>Primers used for the quantitative real-time PCR analysis.</p

Sequence analysis of SmMYB39.

<p>(A) Nucleotide sequence of <i>SmMYB39</i> with amino acid translation. R2 and R3 repeats are highlighted in blue and orange, respectively. (B) Amino acid alignment of SmMYB39 with known R2R3-MYB regulators of phenylpropanes metabolism from other species. R2 and R3 repeats are underlined. The boxed sequences are the potential functional motifs. (C) Phylogenetic tree of SmMYB39 and known R2R3 MYB transcription factors from other plant species. Accession numbers of the proteins in the GenBank database are as follows: ZmMYB42 (NP_001106009.1, <i>Zea mays</i>), TaMYB4 (AEG64799.1, <i>Triticum aestivum</i>), PvMYB4d (AEM17351.1, <i>Panicum virgatum</i>), ZmMYB31 (NP_001105949.1, <i>Zea mays</i>), TaMYB29 (AEV91152.1, <i>Triticum aestivum</i>), EoP1 (ADL18407.1, <i>Eremochloa ophiuroides</i>), GhMYB9 (AAK19619.1, <i>Gossypium hirsutum</i>), GmMYBZ2 (NP_001235092.1, <i>Glycine max</i>), EgMYB1 (CAE09058.1, <i>Eucalyptus gunnii</i>), PttMYB4a (CAD98762.1, <i>Populus tremula</i> × <i>Populus tremuloides</i>), HlMYB7 (CCC14990.1, <i>Humulus lupulus</i>), VvMYB4b (ACN94269.1, <i>Vitis vinifera</i>), SsMYB2 (ABP57083.1, <i>Solenostemon scutellarioides</i>), THM27 (NP_001233975.1, <i>Solanum lycopersicum</i>), PhMYB4 (ADX33331.1, <i>Petunia</i> × <i>hybrida</i>), TfMYB6 (AAS19480.1, <i>Tradescantia fluminensis</i>), PgMYB5 (ABQ51221.1, <i>Picea glauca</i>), AtMYB4 (NP_195574.1, <i>Arabidopsis thaliana</i>), MdMYB16 (ADL36756.1, <i>Malus</i> × <i>domestica</i>), PaMYBR (ADY15315.1, <i>Prunus avium</i>).</p

Relative quantitative analysis of <i>SmMYB39</i> expression in transgenic lines and controls of <i>S. miltiorrhiza</i>.

<p>The results were analyzed using the comparative Ct method and presented as fold-changes compared with the control sample (untransformed control). The <i>S. miltiorrhiza actin</i> gene was used as an internal control to normalize expression levels. control: untransformed plant; ox-VC, RNAi-VC: empty vector controls of <i>SmMYB39</i>-overexpressing lines and <i>SmMYB39</i>-RNAi lines; ox-3, ox-17, ox-28: <i>SmMYB39</i>-overexpressing lines; RNAi-11, RNAi-19, RNAi-25: <i>SmMYB39</i>-RNAi lines.</p

Relative expression levels of <i>SmMYB39</i> (A) and total phenolics content (B) in different tissues of <i>S. miltiorrhiza</i>.

<p>The results were analyzed using the comparative Ct method and presented as fold-changes compared with the root. The <i>S. miltiorrhiza actin</i> gene was used as an internal control to normalize expression levels. Data presented here are the mean of three replicates with error bars indicating ± SD.</p

Analysis of related phenolic compounds contents in transgenic lines and controls of <i>S. miltiorrhiza</i>.

<p>Data presented here are the mean of three replicates with error bars indicating ± SD. The asterisks indicate statistically significant differences (<i>P</i><<i>0.05</i>) compared to the empty vector control. control: untransformed plant; ox-VC, RNAi-VC: empty vector controls of <i>SmMYB39</i>-overexpressing lines and <i>SmMYB39</i>-RNAi lines; ox-3, ox-17, ox-28: <i>SmMYB39</i>-overexpressing lines; RNAi-11, RNAi-19, RNAi-25: <i>SmMYB39</i>-RNAi lines.</p

Phenolic acids biosynthetic pathway in <i>S. miltiorrhiza</i>.

<p>Multiple enzymatic steps are represented by dotted lines. The ‘circle’ and ‘square’ are used to distinguish between the downstream pathway of RA and other branches of phenylpropanes metabolism. C3H, coumarate 3-hydroxylase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumaric acid CoA-ligase; COMT, caffeic acid O-methyltransferase; HPPR, 4-hydroxyphenylpyruvate reductase; PAL, phenylalanine ammonia-lyase; RAS, rosmarinic acid synthase; TAT, tyrosine aminotransferase.</p