SmbHLH53 is relevant to jasmonate signaling and plays dual roles in regulating the genes for enzymes in the pathway for salvianolic acid B biosynthesis in Salvia miltiorrhiza

Highlights

• SmbHLH53 dramatically responds to wounding and multiple hormones response including MeJA, GA3, and ABA treatment.

• SmbHLH53 forms a homodimer as well as a heterodimer with SmbHLH37.

• SmbHLH53 interacts with both SmJAZs1/3/8 and SmMYC2, the core members of the JA signal pathway.

• SmbHLH53 represses the expression of SmTAT1 while simultaneously activating Sm4CL9.

Abstract

Basic helix-loop-helix (bHLH) transcription factors play essential roles in myriad regulatory processes, including secondary metabolism. In this study with Salvia miltiorrhiza, we isolated and characterized SmbHLH53, which encodes a bHLH family member. Expression of this gene was significantly induced by wounding and multiple hormones, including methyl jasmonic acid; transcript levels were highest in the leaves and roots. Phylogenetic analysis indicated that SmbHLH53 clusters with AtbHLH17 and AtbHLH13, two negative regulators of jasmonate (JA) responses, and is localized in the nucleus and cell membrane. Yeast two-hybrid and bimolecular fluorescent complementation assays indicated that SmbHLH53 forms a homodimer as well as a heterodimer with SmbHLH37. It also interacts with both SmJAZs1/3/8 and SmMYC2, the core members of the JA signal pathway. Unexpectedly, we noted that overexpression of SmbHLH53 did not significantly influence the concentrations of rosmarinic acid and salvianolic acid B in transgenic plants. Results from yeast one-hybrid assays showed that SmbHLH53 binds to the promoters of SmTAT1, SmPAL1, and Sm4CL9, the key genes for enzymes in the pathway for phenolic acid synthesis. Assays of transient transcriptional activity demonstrated that SmbHLH53 represses the promoter of SmTAT1 while activating the promoter of Sm4CL9. Thus, the present work revealed that SmbHLH53 may play dual roles in regulating the genes for enzymes in the pathway for Sal B biosynthesis.

Introduction

Salvia miltiorrhiza Bunge, a perennial medicinal herb within Labiatae, is considered a model medicinal plant (Li and Lu, 2014). Its dry roots and rhizomes, commonly known as ‘Danshen’ in China, are some of the most frequently utilized materials in traditional Chinese medicine. Danshen was first recorded in the Shen Nong's Herbal Classic and has been used for more than 2,000 years to treat ailments such as cardiovascular and coronary heart diseases, dysmenorrhea, and amenorrhea (Cheng, 2006, Liu et al., 2016, Zhang et al., 2019). Results from chemical and pharmacological studies have indicated that the main bioactive components in S. miltiorrhiza are lipid-soluble tanshinones and water-soluble salvianolic acids, including salvianolic acid B (Sal B) and rosmarinic acid (RA) (Zhou et al., 2005, Gao et al., 2012). In view of increased clinical demands but a low yield of the active ingredients from this species, researchers have adopted several strategies to augment the accumulation of these active components, including the application of biotic elicitors (Yu et al., 2019), engineering of enzyme genes in the biosynthetic pathway (Wei et al., 2019), and ectopic expression of transcription factors (TFs) (Yang et al., 1804, Li et al., 2018, Huang et al., 2019, Sun et al., 2019).

The endogenous phytohormone jasmonate (JA) plays an essential role in various processes for plant defenses and development. When exposed to external stress, plants begin to accumulate JA and activate the JA signal pathway, resulting in activation of a subset of immune genes and the production of defensive secondary metabolites (Campos et al., 2014). Exogenous application of methyl jasmonic acid (MeJA) on plants has been widely used to stimulate the manufacture of secondary metabolites, including terpenoids, flavonoids, alkaloids, and phenylpropanoids (Wasternack and Strnad, 2019). In S. miltiorrhiza, both lipid-soluble tanshinones and water-soluble salvianolic acids are significantly increased upon treatment with MeJA (Ge et al., 2015, Hao et al., 2015). The molecular mechanism by which it promotes the accumulation of valuable active ingredients in S. miltiorrhiza has already been partially elucidated (Shi et al., 2016, Zhou et al., 2016, Yang et al., 1804, Du et al., 2018, Pei et al., 2018). For example, in response to MeJA treatment, JA signaling is activated in such plants, which then leads to degradation of jasmonate ZIM-domain (JAZ) proteins and de-repression of SmMYC2. The latter promotes the production of RA and Sal B by binding to and activating the promoters of SmTAT1, SmPAL1, and SmCYP98A14, three key enzyme genes in the pathway for phenolic acid synthesis (Yang et al., 1804, Du et al., 2018).

Basic helix-loop-helix (bHLH) proteins, one of the largest TF families in plants, regulate various physiological or morphological events, including iron metabolism, hormone synthesis, root development, abiotic stress tolerance, synthesis of secondary metabolites, flowering, and fruit maturation (An et al., 2019). The bHLH family comprises an HLH domain that consists of 50 to 60 amino acids, as well as a highly conserved HER motif (His-Glu-Arg) in the N-terminal (Pires and Dolan, 2010). The HLH domain is evolutionarily conserved and promotes the formation of homo- or heterodimers (Buck and Atchley, 2003, Goossens et al., 2017). The N-terminal is associated with its binding to DNA sequences, allowing the bHLH protein to adhere specifically to either the E-box (5′-CANNTG-3′) or the G-box (5′-CACGTG-3′) (Feller et al., 2011, Sun et al., 2015, Kavas et al., 2016, Mao et al., 2017). This bHLH family has been divided into 26 subfamilies according to the presence of highly conserved short amino acid (aa) motifs (Pires and Dolan, 2010). The bHLHs from subfamilies IIIe, IIIf, or IIId have been widely reported to modulate the biosynthesis of secondary metabolites. As a typical member of the IIIe subfamily, MYC2 positively or negatively regulates secondary metabolism during JA signaling in a species-specific manner (Dombrecht et al., 2007, Todd et al., 2010, Zhang et al., 2011). Most bHLHs related to flavonoid biosynthesis have been assigned to subfamily IIIf (Zhao et al., 2019). In Arabidopsis thaliana, bHLH17, bHLH13, and bHLH3 -- all within the IIId subfamily -- interact with JAZ proteins and negatively control JA responses, including the accumulation of anthocyanin (Fonseca et al., 2014). In particular, they antagonize MYC2, MYC3, and MYC4 and redundantly reduce those responses (Nakata et al., 2013).

Based on genome-wide analyses, 127 bHLH genes have been identified in S. miltiorrhiza (Zhang et al., 2015). Some of those regulate the biosynthesis of phenolic acids or tanshinones in that species. For example, SmbHLH51, within the IIIf subfamily, is a positive transcriptional regulator of the pathway for phenolic acid biosynthesis (Li et al., 2018); SmbHLH10 positively regulates tanshinone biosynthesis in hairy roots (Xing et al., 2018a); SmbHLH148 up-regulates the production of both phenolic acids and tanshinones by activating the biosynthetic pathways of two groups of pharmaceutical ingredients (Xing et al., 2018b); and SmbHLH3 negatively regulates the accumulation of both phenolic acids and tanshinones by directly suppressing the transcription of key enzyme genes (Zhang et al., 2020). We previously reported that overexpression of SmMYC2 increases the production of RA and Sal B in S. miltiorrhiza (Yang et al., 2017), and that SmbHLH37 (IIId subfamily) antagonizes the transcription activator SmMYC2 in controlling RA and Sal B biosynthesis (Du et al., 2018). However, for most of those 127 bHLHs, little is known about their roles in that species.

SmbHLH53 is thought to be involved in regulating secondary metabolism (Zhang et al., 2015). However, because details about its characterization and function have not previously been reported, we focused on this gene for its responsiveness to JA signaling. Within that pathway SmbHLH53 can form either a homodimer or heterodimer and interact with both SmJAZs1/3/8 and SmMYC2, two core members. Therefore, we speculated that SmbHLH53 participates in the regulation of RA and Sal B biosynthesis. Unexpectedly, we found that overexpression of SmbHLH53 does not significantly influence the concentrations of those phenolic acids in S. miltiorrhiza. To elucidate the responsible molecular mechanism, we used yeast one-hybrid (Y1H) assays and monitored transient transcriptional activity (TTA) for further investigations of the relationships between SmbHLH53 and genes for enzymes in the pathway for Sal B biosynthesis.