Biosynthesis and signal transduction of plant growth regulators and their effects on bioactive compound production in Salvia miltiorrhiza (Danshen)

Plant growth regulators (PGRs) are involved in multiple aspects of plant life, including plant growth, development, and response to environmental stimuli. They are also vital for the formation of secondary metabolites in various plants. Salvia miltiorrhiza is a famous herbal medicine and has been used commonly for > 2000 years in China, as well as widely used in many other countries. S. miltiorrhiza is extensively used to treat cardiovascular and cerebrovascular diseases in clinical practices and has specific merit against various diseases. Owing to its outstanding medicinal and commercial potential, S. miltiorrhiza has been extensively investigated as an ideal model system for medicinal plant biology. Tanshinones and phenolic acids are primary pharmacological constituents of S. miltiorrhiza. As the growing market for S. miltiorrhiza, the enhancement of its bioactive compounds has become a research hotspot. S. miltiorrhiza exhibits a significant response to various PGRs in the production of phenolic acids and tanshinones. Here, we briefly review the biosynthesis and signal transduction of PGRs in plants. The effects and mechanisms of PGRs on bioactive compound production in S. miltiorrhiza are systematically summarized and future research is discussed. This article provides a scientific basis for further research, cultivation, and metabolic engineering in S. miltiorrhiza.

The dried roots and rhizomes of S. miltiorrhiza act as medicinal parts, which are brownish red or brick red in surface color, white inner surface.Until now, over 200 substances have been discovered and isolated from S. miltiorrhiza [50].The primary pharmacological components of this plant are hydrophilic phenolic substances and lipophilic diterpenoid quinones, which are produced through secondary metabolic pathways.Hydrophilic phenolic substances, including rosmarinic acid, caffeic acid, salvianolic acid A, salvianolic acid B and are produced via phenylpropanoid and tyrosine-derived pathways [82,83] (Fig. 4a).Salvianolic acid B, one of the quality control indicators of S. miltiorrhiza in Chinese pharmacopeia and United States Pharmacopoeia, is mainly distributed in the phloem and xylem of S. miltiorrhiza roots [84].Some key enzymes, such as phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl CoA ligase (4CL), tyrosine aminotransferase (TAT), p-hydroxyphenylpyruvate reductase (HPPR), rosmarinic acid synthase (RAS), and CYP98A14, are involved in the pathways [81,82].For rosmarinic acid, although its biosynthetic Fig. 2 The geographical distribution of S. miltiorrhiza.Data were obtained from the Global Biodiversity Information Facility (https:// www.gbif.org/) pathway is still not fully understood, four biosynthetic pathways have been proposed for its formation, including the synthesis of rosmarinic acid from caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid (Way 1), the caffeoyl-4′-hydroxyphenyllactic acid branch (Way 2), the 4-coumaroyl-4′-hydroxyphenyllactic acid branch (Way 3), and the 4-coumaroyl-3′,4′-dihydroxyphenyllactic acid branch (Way 4) [83,85] (Fig. 4a).Lipophilic diterpenoid quinones, the focus of tanshinone analogues, including dihydrotanshnone I, tanshinone I, tanshinone IIA, and cryptotanshinone, originate from the 2-C-methyld-erythritol-4-phosphate (MEP) pathway and/or the mevalonate (MVA) pathway [86,87] (Fig. 4b).The main processes in the MEP pathway and/or the MVA pathway include isoprene precursor synthesis, direct skeleton precursor production, and tanshinones formation [88].All terpenes go through the first two processes, but terpenoid synthases and modifying enzymes, which can vary depending on the species, are involved in the latter [88].According to previous report, tanshinones are mostly present in the periderm [80].It is generally believed that the root of S. miltiorrhiza with deep crimson-colored brown and extensive branches has a higher content of tanshinone.Some key enzymes, including acetyl-CoA C-acetyltransferase (AACT), hydroxymethylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose5-phosphate reductoisomerase (DXR), isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranylgeranyl diphosphate synthase (GGPPS), copalyl diphosphate synthase (CPS), kaurene synthase-like (KSL), cytochrome P450 monooxygenases (CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, CYP71D375), and 2-oxoglutarate-dependent dioxygenase 3 (2OGD3 or 2-ODD3), along with other unknown enzymes, are associated with the tanshinone biosynthetic pathway [80,[89][90][91][92][93][94][95] (Fig. 4b).Additionally, polysaccharides and other secondary metabolites, such as monoterpenes, sesquiterpenes, triterpenes, sterols, flavonoids, anthocyanidins, proanthocyanidins, alkaloids, and quinones, play important roles in medicines [54,81,[96][97][98][99][100].A previous study reported that salvinolic acids and tanshinones made up most of the active compounds found in roots, whereas stachyose made up the majority of saccharides; except for tanshinones, salvianolic acids, flavonoids, and triterpenes were found in the leaves, stems, and flowers, and the majority of the saccharides were monosaccharides [101].Based on traditional Chinese medicine and the active ingredients present, several commercial medicines derived from S. miltiorrhiza have been developed in China.They include Danshen injection, Danshensu sodium injection, salvianolic acid B injection, Danhong injection, tanshinone IIA sodium sulfonate injection, salvia polyphenolic acid salt injection, compound Danshen tablets, and compound Danshen dripping pills [71,102].In addition, healthcare products such as Danshen flower tea, Danshen leaf tea, and cosmetics contained Danshen extract have also been developed in China.We believe that S. miltiorrhiza will have a broader application in food, medicine, cosmetics, healthcare products and other In S. miltiorrhiza, although there are limited references to the regulatory roles of PGRs on plant growth and development, significant advancements have been made in understanding the regulatory roles of PGRs in the secondary metabolite accumulation.In this article, to better understand the mechanism of action of PGRs on active compound production of S. miltiorrhiza, the biosynthesis and signal transduction pathways of PGRs in plants are briefly reviewed.Then, the effects of PGRs on bioactive compound production, mainly phenolic acids and tanshinones, and the underlying mechanisms in S. miltiorrhiza, are systematically summarized.Future research perspectives are also discussed.This article provides important implications for further research into the function and regulatory mechanisms of PGRs in the active compound production, field cultivation and metabolic engineering of S. miltiorrhiza.

Gibberellins
GAs are produced in plastids through the MEP pathway from geranylgeranyl diphosphate (GGPP).GGPP is primarily formed from pyruvate and glyceraldehyde 3-phosphate (G3P) (Fig. 5).GGPP formation also requires crosstalk between the MEP and MVA pathways in the cytoplasm during diterpene biosynthesis [114].GAs are produced in various plant parts, including the root and stem apical meristems, developing leaves, and seed embryos.GA 1 , GA 3 , GA 4 , and GA 7 are the primary bioactive GAs.GA 4 and GA 7 , occur naturally, GA 3 is synthetic, and GA 1 can be synthesized in plants and fungi [115].The major elements of GA signaling in Arabidopsis consist of the gibberellin-insensitive dwarf1 (GID1)-GA receptor, the DELLA proteins (DELLAs)-growth repressor, and the F-box protein SLEEPY1 (SLY1) [116] (Fig. 1).DELLAs are target elements of the signaling system and are considered to combine signals from different hormones.

Salicylic acid
SA, originally derived from the leaves and bark of willow trees, is a phenolic phytohormone that can be produced via the isochorismate synthase (ICS) pathway and PAL pathway, both of which require chorismate as a precursor [29,125].It contains active analogs, including SA, acetyl SA, methyl SA, and dihydroxy benzoic acid [126].SA signaling is not a simple linear pathway.Currently, the exact mechanism underlying SA signaling is still poorly understood [127].Non-expressors of PR genes (NPRs), including as NPR1, NPR3, and NPR4, which function as authentic SA receptors, are the most well-characterized SA-binding proteins and directly interact with the TGA transcription factors [128,129] (Fig. 1).

Strigolactones and karrikins
SLs and KARs are butenolides.SL biosynthesis primarily synthetize in the roots and is initiated in the plastids by the isomerase DWARF27 (D27).This enzyme converts all trans-β-carotene to 9-cis-β-carotene.Sequentially, carotenoid cleavage dioxygenase 7 (CCD7) and CCD8 act on this substrate to produce carlactone (CL).CL is catalyzed by the CYP711A subfamily of cytochrome P450 (CYP450) enzymes, eventually producing a different series of SLs [130] (Fig. 5).GR24 is a synthetic analog of SLs that demonstrates higher potency and stability compared with natural SLs.It is widely used and serves as a standard and model compound for studying the characteristics and functions of SLs in plant physiology due to its accessibility and universality [131,132].SL signaling is mainly mediated by DWARF14 (D14), MAX2, and the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, which degrades the transcriptional repressors SMXL6/7/8 (suppressor of max2 1 (smax1)-like 6/7/8) in Arabidopsis [32] (Fig. 1).KARs impact various facets of plant growth and development.They are originally identified to be seed germination stimulators in post-fire species after being released from burning plant debris [133].There might be similarities between the processes of SL and KAR signaling.KAI2, a paralog of SL receptor D14, is widely recognized as a receptor for exogenous KARs and unidentified endogenous KLs.It binds to the F-box protein MAX2 to target SMAX1 and SMXL2 in Arabidopsis [134].

Nitric oxide
NO is a crucial compound for gas signaling.Sodium nitroprusside (SNP) is often regarded as a NO donor and has been used to investigate its diverse biological regulatory functions [135].In plants, NO biosynthesis is a complicated process and can occur via a variety of oxidative and reductive pathways [3,39].However, it is unclear what metabolic processes underlie variations in NO levels in plants.NO signaling appears to be mediated primarily by secondary messengers, including cyclic guanosine monophosphate (cGMP), cyclic ADP-ribose (cADPR), and Ca 2+ mobilization [9] (Fig. 1); however, the specific mechanism is not clear.

Polyamines
PAs are a class of aliphatic nitrogenous bases with low molecular weight that are present in nearly every live cell, such as putrescine (Put), spermidine (Spd), and spermine (Spm) that are the most prevalent compounds [136].Almost all free-living eukaryotes synthesize Put directly from ornithine, which is then catalyzed by spermidine synthase and spermine synthase to form Spd and Spm.They possess an additional biosynthetic pathway for Put formation via the arginine pathway, which involves arginine decarboxylase, agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase [137].PA signaling is associated with the direct interaction of various metabolic processes and complex hormonal crosstalk, integrated with the processes of reactive oxygen species (ROS) signaling, NO production, the regulation of ion channel activity, and Ca 2+ homeostasis [138].

Effects of PGRs on active compound production in S. miltiorrhiza
It is highly interesting to explore the active pharmacological compounds in medicinal plants.PGRs have a major impact on how medicinal plants produce secondary metabolites [139].Significant reduction of platycogenintype saponins has been found in platycodon roots treated with paclobutrazol [140].In S. miltiorrhiza, some PGRs also regulate the active compound production (Table 2).For example, 10 μΜ of IAA considerably enhanced the formation of lateral roots and the production of cryptotanshinone, tanshinone I, and tanshinone IIA [141].TDZ (0.1 or 0.5 mg/L) has demonstrated a notable improvement in the survival rate of S. miltiorrhiza regenerated plantlets.The extracts of the roots of these regenerated plantlets contained salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA [142].After treatment with N 6 -benzyladenine (0.2 mg/L, the same chemical as BAP), the level of cryptotanshinone in S. miltiorrhiza calli reached the maximum [143].Treatment with 50 mg/L of GA has been found to significantly increase the dihydrotanshinone I, tanshinone I, and tanshinone IIA contents.In contrast, treatment with 100 μΜ of GA 3 significantly improved the content of salvianolic acid B, rosmarinic acid, dihydrotanshinone  I, cryptotanshinone, tanshinone I, and tanshinone IIA in hairy roots of S. miltiorrhiza [144,145].A previous study has indicated that 10-150 μΜ of GA 3 only enhanced the level of salvianolic acid B and rosmarinic acid in hairy roots of S. miltiorrhiza [146].The reasons for these differences are worth investigating further.Moreover, 50-200 μΜ of ABA promoted the production of salvianolic acid A, salvianolic acid B, rosmarinic acid, caffeic acid, dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA in S. miltiorrhiza hairy roots [147][148][149][150]. Additionally, it induced the transcription of numerous differentially expressed genes (DEGs) associated with ABA biosynthesis and signal transduction [151].It appears that low concentrations of ABA are more likely to regulate the accumulation of phenolic acids, whereas high concentrations of ABA are more likely to regulate the accumulation of tanshinones.However, the regulatory mechanism is largely unknown.By applying 0.05-0.50mM of ET to S. miltiorrhiza calli for 60 days and 200 μg/L of ET to hairy roots for eight days, a notable rise in the quantity of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA has been found [144,152], and 170 AP2/ERF genes have been identified [153].In S. miltiorrhiza hairy roots, treatment with 50 or 100 μM of ET for 6 days has also positively regulated the level of salvianolic acid B, rosmarinic acid, and caffeic acid [146].A recent report revealed that adding 70 μM of ET to S. miltiorrhiza hairy roots for 50 days inhibited the amount of dihydrotanshinone, cryptotanshinone, and tanshinone I [154].The various effects of ET on the active compounds could mainly be attributed to differences in the plant culture system, concentration, and duration of ET.
Different MeJA treatments have been demonstrated to greatly raise the level of phenolic acids and/or tanshinones in the hairy roots and calli of S. miltiorrhiza.The synthesis of phenolic acids in hairy roots of S. miltiorrhiza is more efficiently improved than that of tanshinones [155][156][157][158][159][160][161][162][163].Compared with other PGRs, the effects and regulatory mechanisms of JAs on the active compound production of S. miltiorrhiza have been extensively studied.SA has been reported to induce the synthesis of salvianolic acid B, rosmarinic acid, and caffeic acid in S. miltiorrhiza hairy roots and cell cultures in concentration-and time-dependent manners [164][165][166].The accumulation of these compounds leads to the induction of many DEGs involved in SA signaling, antioxidant systems, hormone biosynthesis and signaling, defenserelated CYP450, and ATP-binding cassette (ABC) [167].In addition, SA has been reported to increase the level of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA; however, their yields were relatively low [168,169].To date, only one report of SL in S. miltiorrhiza has been published.The study revealed that under arbuscular mycorrhizae and GR24 treatment, the maximum amount of tanshinone IIA and cryptotanshinone production was shown in (NH 4 ) 2 SO 4 -treated S. miltiorrhiza roots [170].SNP significantly stimulated tanshinone production and increased cryptotanshinone and tanshinone IIA accumulation [156].It is possible that NO signaling is crucial to the production of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA triggered by water deficiency in S. miltiorrhiza hairy roots, mainly through stimulation of the MEP pathway [171].Both ABA and PAs promote salvianolic acid synthesis in the S. miltiorrhiza hairy roots and that Put and Spd are superior to Spm in promoting salvianolic acid B and salvianic acid A synthesis [147].These studies suggest that PGRs effectively enhance the accumulation of salvianolic acids and/or tanshinones in S. miltiorrhiza.Moreover, the yield of salvianolic acids and/or tanshinones is influenced by the type, dosage, and application time of the PGRs, as well as the plant cultivation system.However, the specific mechanisms involved require further investigation.

Mechanisms by which PGRs regulate active compound production in S. miltiorrhiza
According to the previous studies, PGRs regulate the physiological, biochemical, and transcriptional levels of active compounds in S. miltiorrhiza.Furthmore, PGRs play a vital role in the active compound production of S. miltiorrhiza through transcription factors, enzymes, enzyme-encoding genes, signal transduction, post-transcriptional regulation, and crosstalk.

PGRs regulate active compound production in S. miltiorrhiza through transcription factors
Considerable advancements have been achieved in studying the roles and regulatory mechanisms of PGRs in the secondary metabolites of S. miltiorrhiza associated with transcription factors (Fig. 7).For example, GA-responsive SmGRAS1/2 plays an inhibitory role in root growth and the biosynthesis of rosmarinic acid and salvianolic acid (See figure on next page.)Fig. 7 The PGRs-responsive transcription factors and related genes in S. miltiorrhiza.Light blue circle represents the PGRs.Yellow circle represents the transcription factors.Blue circle represents phenolic acid biosynthesis genes.Red circle represents tanshinone biosynthesis genes.The networks were visualized using Cytoscape software (v.3.10.2) (https:// cytos cape.org/) B but it promotes the production of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by binding to the SmKSL1 promoter in S. miltiorrhiza hairy roots [172].In contrast, SmGRAS3 promotes the biosynthesis of cryptotanshinone, dihydrotanshinone I, and tanshinone I [173].Some transcription factors that respond to ABA also have an important effect on the active compound production of S. miltiorrhiza.For instance, the SmWRKY34-SmbZIP3 negatively regulates the production of rosmarinic acid, salvianolic acid B, cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by interacting with SmTAT and two transcription factors, SmERF128 and SmMYB9b that associated with tanshinone biosynthesis [174].SmbZIP1 positively promotes the biosynthesis of rosmarinic acid and salvianolic acid B by enhancing the expression of biosynthetic genes, such as SmC4H1, while decreasing the level of cryptotanshinone, dihydrotanshinone, tanshinone I, and tanshinone IIA, mainly by suppressing the expression of biosynthetic genes, such as SmGGPPS [175].SmbZIP2 reduced the amount of rosmarinic acid and salvianolic acid B by linking to the SmPAL promoter and physically interacts with several SnRK2s, including SnRK2.3,SnRK2.4,SnRK2.6, and SnRK2.10 [176].SmHD-Zip12 positively regulates the level of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by altering the transcription of some key enzyme-coding genes, such as SmAACT , SmDXS, SmGGPPS, SmCPS1, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 [177].
In general, PGRs regulate active compound production in S. miltiorrhiza through transcription factors directly binding to the promoters of genes related to the active compound biosynthesis.These genes include key enzyme-encoding genes of the biosynthetic pathway of active compounds, or other transcription factors.However, the effects and mechanisms of these PGRs on the transcription factors need to be further investigated.

PGRs regulate active compound production in S. miltiorrhiza through enzymes or enzyme-encoding genes
In S. miltiorrhiza, PGRs also regulate secondary metabolites by modifying the activity of key enzymes or expression of key enzyme-encoding genes of secondary metabolic biosynthetic pathways.For example, exogenous IAA significantly promote the concentration of tanshinone and stimulate the transcription of several key enzyme-encoding genes involved in the tanshinone biosynthesis pathway, such as SmCPS1, SmDXR, SmDXS2, and SmKSL1 [141].Both GA 3 and ET have been found to be effective in promoting phenolic acid accumulation and increasing PAL and TAT activities in the hairy roots of S. miltiorrhiza [146].GAs and ET treatments also improve the transcription of SmKSL and SmCPS1, leading to tanshinone accumulation [144,145,152].Most recently, ET was found to decrease the transcription of SmCPS1, SmGGPPS, and SmCYP76AH1 as well as the production of tanshinones [150].MeJA [155-157, 160, 161, 168, 188-191], ABA [146-151, 188, 191], and SA [165,166,168,169,[188][189][190][191][192][193] have been shown to induce the transcription of some genes linked to phenolic acids and tanshinones biosynthesis, such as SmPAL, SmC4H, SmRAS, SmTAT , SmHMGR, SmDXS, SmDXR, SmCPS, SmGGPPs, and SmKSL.In addition, both MeJA and SA regulate the transcription of 4-hydroxyphenylpyruvate dioxygenase (Smhppd), which significantly contributes to the accumulation of rosmarinic acid in S. miltiorrhiza [194].Transcriptional data analysis has suggested that MeJA improves the transcription of some genes linked to tanshinone and phenolic acid biosynthesis, such as the 2C methyl-D-erythritol 2,4-cyclodiphosphate synthase (SmMEC or SmMDS) and the caffeoyl-CoA O-methyltransferase (SmCCoAOMT) genes.The transcription of these genes had a significant impact on the level of tanshinones and phenolic acids in S. miltiorrhiza [163,195,196].NO has been found to promote tanshinone accumulation and enhance SmHMGR and SmDXR expression [156,171].
In S. miltiorrhiza, some genes related to the PGRs biosynthetic pathway regulate secondary metabolites.For example, in the JAs biosynthetic pathway, overexpression of SmAOC positively regulates the biosynthesis of tanshinone IIA, rosmarinic acid, and lithospermic acid B in S. miltiorrhiza hairy roots [197].Overexpression of SmJMT increases the level of salvianolic B and rosmarinic acid by simultaneously activating the expression of SmPAL1, SmC4H, Sm4CL3, SmTAT , SmHPPR, SmRAS, and SmCYP98A and raising the endogenous levels of MeJA [198].In S. miltiorrhiza, 23 potential JA biosynthesis-related genes have been reported, including nine SmLOXs, seven SmAOSs, two SmAOCs, and five SmOPR3s [199].MeJA has been proven to be effective in the accumulation of salvianolic acids and/or tanshinones in S. miltiorrhiza, and one of the SmAOCs related to the production of tanshinones and phenolic acids has been previously published [196]; therefore, these JA biosynthetic genes may play vital roles in the accumulation of tanshinones and/or phenolic acids.GA may also affect tanshinones production by changing the transcription of some genes including ent-copalyl diphosphate synthase (SmCPS), ent-kaurene synthase (SmKS) and entkaurene oxidase (SmKO), which encode enzymes in the biosynthetic pathway of GA in S. miltiorrhiza [200,201].Twenty-two candidate genes of GA metabolism pathway have been systematically identified, including one SmKO, two SmKAOs, six SmGA20oxs, two SmGA3oxs, and 11 SmGA2oxs.These genes play significant roles in GA production and the interaction between GA accumulation and tanshinone production in S. miltiorrhiza [114].
Taken together, PGRs directly modulate the activity of key enzymes or expression of key enzyme-encoding genes of active compound biosynthetic pathways, or indirectly influence the activity of key enzymes or expression of key enzyme-encoding genes by the transcription of PGRs biosynthetic pathway genes or other protein genes, to regulate active compound production in S. miltiorrhiza.However, little is known about how these PGRs regulate the enzyme activity and enzyme-encoding gene expression.

PGRs regulate active compound production in S. miltiorrhiza via signal transduction
By influencing the transcription of genes associated with PGR signal transduction, PGRs also regulate secondary metabolites of S. miltiorrhiza.For example, exogenous IAA significantly promotes tanshinone content and alters the transcription of AUX biosynthesis-and signal transduction-related genes, including AUX/IAAs, GH3s, and SAURs [141].Genome-wide investigation has revealed 23 AUX/IAA genes and 25 ARF genes in S. miltiorrhiza [206,207].These genes may be important for the growth and development of plants as well as the final content of secondary metabolites in S. miltiorrhiza.Moreover, ABA signaling genes have been found to modulate the biosynthesis of phenolic acids and tanshinones.For instance, SmSnRK2.6, which is linked to ABA signaling, acts as an inducer in the production of rosmarinic acid and salvianolic acid B through interactions with SmAREB1 in the hairy roots of S. miltiorrhiza [208].Recently, Ding et al. have reported that SA regulates the level of salvianolic acid B and rosmarinic acid via the SmNPR1-SmTGA2/ SmNPR4 or SmNPR4-SmTGA5 modules in S. miltiorrhiza hairy roots [209,210].SA also has been shown to significantly promote rosmarinic acid accumulation through secondary messengers associated with signal transduction, including H 2 O 2 , NO, and Ca 2+ [192,193,211].
Several ERFs associated with ET signaling pathway, have been proven to modulate the level of phenolic acids and tanshinones (Fig. 7).ET-sensitive SmERF6/8/128/SmERF1b-like proteins can enhance the biosynthesis of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by regulating the gene expression of SmCPS1, SmKSL1, and SmCYP76AH1 from MEP pathway [223][224][225][226]. Conversely, overexpression of the ET-responsive transcription factor EIN3-like 1 (SmEIL1) suppresses tanshinone accumulation by inhibiting the transcription of the SmCPS1 [153].Furthermore, some genes linked to signal transduction and the level of tanshinones and phenolic acids in S. miltiorrhiza, also respond to PGRs.For example, SmMAPK3 regulates the level of rosmarinic acid and salvianolic acid B via the SmMAPKK2/4/5/7-SmMAPK3-SmJAZ cascade, in response to SA and MeJA [227].The expression of SmGH3.2,SmGH3.6, and SmGH3.10,which are involved in the AUX response, is up-regulated by MeJA in S. miltiorrhiza [228].Several U-box E3 (UBE3) genes may be linked with the accumulation of phenolic acids or tanshinones via the ABA signaling pathway [229].However, to fully understand the regulatory mechanisms of these genes, further studies are needed.
So far, for this regard, the signal transduction of JAs, SA, ABA, and NO that regulate active compound production in S. miltiorrhiza is relatively clear.The regulatory mechanism of other PGRs including SLs, KLs, BR, PAs in active compound production in S. miltiorrhiza, is still little known.In our opinion, to gain a lot of knowledge of the regulatory mechanism of PGRs in active compound production in S. miltiorrhiza via signal transduction, identification of the biosynthesis and signaling transduction pathways of PGRs in S. miltiorrhiza should be firstly paid close attention.

PGRs regulate active compound production in S. miltiorrhiza via post-transcriptional regulation
MicroRNAs (miRNAs) act as important regulators of plant development, secondary metabolism, and environmental stimuli through cleavage or translational inhibition [230,231].By targeting genes that encode enzymes and transcription factors associated with biosynthetic pathways, miRNAs influence a variety of bioactive compound production, including terpenoids, alkaloids, flavonoids, and phenolic acids [232].It has been discovered that Mdm-miR858 targets MdMYB9 and MdMYBPA1 to take part in the production of anthocyanins in red-fleshed apple plants [233].miR828 and miR858 have been found to target VvMYB114 to promote the production of anthocyanin and flavonols in grapes [234].Many miRNAs have been identified in S. miltiorrhiza [232], some of which are known to be responsive to PGRs.For example, miR396b, activated by MeJA, ABA, GA, etc., can function as an important upstream regulator of cell growth and tanshinones and salvianolic acids accumulation in S. miltiorrhiza [235].Overexpression of MIR160a might negatively regulate tanshinones biosynthesis by increasing IAA accumulation and decreasing SA and JA levels [236].Smi-miR12112 is responsive to MeJA treatment and potentially affects phenolic acid biosynthesis by targeting SmPPOs [230].However, the specific regulatory effects of PGR-responsive miRNAs on secondary metabolites accumulation in S. miltiorrhiza remain unclear.In S. miltiorrhiza, the function of a great many miRNAs is unknown and the response of miRNAs to PGRs is also poorly known, therefore, the study on PGRs regulating active compound production in S. miltiorrhiza via post-transcriptional regulation will have great research prospects.

PGRs regulate active compound production in S. miltiorrhiza through crosstalk
Under both normal and stressful circumstances, PGRs are of vital importance in regulating various aspects of plant growth and development and operate within complex networks (Fig. 1).Studies on a variety of plants have suggested the function of PGR crosstalk in the secondary metabolite accumulation.For example, in in vitro shoot cultures of Bacopa monnieri, MeJA combined with SA has been demonstrated to considerably raise the biomass and active bacoside A content [237].Additionally, in the root suspension of Ajuga bracteosa, the combined action of MeJA and PAA increases the total phenolic and flavonoid contents [238].GR24 combined with ABA delays anthocyanin accumulation and reduces the anthocyanin biosynthetic gene expression in grapevine berries [33].Crosstalk between PGRs has a great impact on active compound accumulation of S. miltiorrhiza (Table 2; Fig. 8).For example, endogenous MeJA accumulation is triggered by exogenous application of polyethylene glycol and ABA, which activate the ABA signaling pathway and improve tanshinone synthesis.In contrast, in S. miltiorrhiza hairy roots, exogenous MeJA might directly trigger tanshinone synthesis mostly through the MEP pathway [147].Through a JA-dependent signaling mechanism, NO promotes the synthesis of tanshinone I triggered by KAR [239].H 2 O 2 and NO elicited by SA can work alone or together to enhance salvianolic acid B accumulation in SA-stimulated S. miltiorrhiza cells [192].JA participates in the signal transduction pathway that drives the yield of salvianolic acid B and tanshinone in response to KAR stimulation in S. miltiorrhiza [239][240][241].ABA, GA, and ET are effective in inducing phenolic acid production and increasing PAL and TAT activities in S. miltiorrhiza hairy roots, and the synthesis of phenolic compounds driven by ABA and ET requires GA signaling [145].
Moreover, some transcription factors regulate active compound production in S. miltiorrhiza by modulating the PGR balance (Fig. 7).For example, SmMYB52 inhibits the expression of key enzyme-encoding genes associated with the IAA biosynthesis and activates key enzyme-encoding genes associated with the JA and salvianolic acid B biosynthesis [242].Overexpression of rSmSPL2 has a negative effect on the level of salvianolic acid B and rosmarinic acid in S. miltiorrhiza roots.However, it significantly increases the endogenous CK levels and dramatically decreases the endogenous AUX levels by binding to the promoters of Sm4CL9, SmTAT1, and SmPAL1 and inhibiting their transcription [243].
In contrast, some transcription factors that influence the active compound production in S. miltiorrhiza have been demonstrated to be responsive to at least two types of PGRs (Fig. 7).Overexpression of SmMYB9b, activated by ABA, GA, and MeJA, results in a higher content of tanshinones by stimulating the MEP pathway [244].SmPAP1, the R2R3-MYB transcription factor, is induced by MeJA, SA, and ABA.By connecting with SmMYC2 and turning on the transcription of SmPAL and SmC4H in the transgenic line roots, it takes part in regulating the biosynthetic pathways of rosmarinic acid, salvianolic acid B, total phenolics, and total flavonoids [245].SmMYB111 responds to SA, GA, and MeJA and serves to positively regulate the rosmarinic acid and salvianolic acid B biosynthetic pathways by interacting with SmTTG1 and SmbHLH51 [246].Both SmSPL6 and SmSPL7 respond to treatment with IAA, GA, MeJA, and ABA.The former directly connects with Sm4CL9 and SmCYP98A14, promoting the yield of rosmarinic acid and salvianolic acid B, whereas the latter binds to the promoters of Sm4CL9 and SmTAT1, inhibiting salvianolic acid B biosynthesis [247,248].ABA induces SmbHLH148 at high levels, whereas MeJA and GA induce it at a moderate level.By inducing the transcription of key enzyme-encoding genes linked to the active compound biosynthesis, such as SmHPPR, SmRAS, SmCYP98A14, SmGGPPS, SmCPS1, SmCPS5, SmKSL1, and SmCYP76AH1, its overexpression improves the level of dihydrotanshinone I, cryptotanshinone, tanshinone I, caffeic acid, rosmarinic acid, and salvianolic acid B in hairy roots of S. miltiorrhiza  [249].SmbHLH53 is responsive to IAA, ABA, GA 3 , and MeJA.It forms a homodimer and heterodimer with Smb-HLH37.By binding to the SmTAT1/PAL1/4CL9 promoter, it inhibits JA signaling, antagonizes SmMYC2, and regulates the content of salvianolic acid B [219,250].SmERF1L1 is activated by MeJA, yeast extract, SA, and ET.This activation increases the dihydrotanshinone and cryptotanshinone contents and decreases the rosmarinic acid and salvianolic acid A contents by binding to the promoter of SmDXR in S. miltiorrhiza [251].SmWRKY1 is responsive to SA, MeJA, and NO; participates in the regulation of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA biosynthesis; and acts as an inducer by activating SmDXR in the MEP pathway [252].SmWRKY42-like proteins respond significantly to exogenous GA and Eth treatment.It may be crucial in enhancing the medicinal properties of S. miltiorrhiza [253].SmGRAS1-5, which may contribute to the tanshinone accumulation, is regulated by GA and ET signaling in S. miltiorrhiza [143].
Furthermore, some enzyme-encoding genes regulating the level of phenolic acids and tanshinones have been shown to be responsive to more than one type of PGR.SmDXR, an enzyme that is essential for the tanshinone biosynthetic pathway, responds to MeJA and SA [254].SmCYP76AK2 and SmCYP76AK3 could respond to MeJA, SA, which significantly reduces the content of cryptotanshinone, tanshinone IIB, dihydrotanshinone, tanshinone I, and tanshinone IIA in S. miltiorrhiza mutants [255].SmLAC25 is responsive to MeJA, AUX, ABA, and GA stimuli, and its overexpression promotes lignin accumulation and decreases rosmarinic acid and salvianolic acid B concentrations in S. miltiorrhiza [256].
As mentioned above, in S. miltiorrhiza, these PGRs solely influence the level of tanshinones and/or phenolic acids.In addition, crosstalk between these PGRs may also occur in other plants, suggesting that the interaction between these PGRs may be critical for the active compound production in S. miltiorrhiza.Moreover, in S. miltiorrhiza, some PGRs have an effect on the level of phenolic acids and tanshinones.However, investigation into the molecular mechanism behind this crosstalk is necessary.For example, GA 3 has been shown to significantly improve the level of cryptotanshinone, and tanshinone IIA in the roots of the HMGR4-overexpression line, whereas IAA considerably inhibits tanshinone accumulation in the studied S. miltiorrhiza root material [257].In S. miltiorrhiza, both MeJA and SA regulate the production and the transcription of tanshinone biosynthesisgenes in the hairy roots of SmGGPPS-overexpressing lines [167].AUXs, CKs, and ABA enhance the production of cryptotanshinone, tanshinone I, and IIA in the hairy roots of S. miltiorrhiza [258].
Many kinds of hormones coexist in plants, which interact with each other to regulate the life activities of plants.Therefore, the crosstalk between PGRs is more important in the formation of active ingredients in S. miltiorrhiza.In addition, the production of tanshinones and phenolic acids is frequently induced by environmental factors, for example, light intensity and quality had a differential impact on the level of active compounds, red and blue light treatments significantly heighten the level of salvianolic acid B, and tanshinone content was more susceptible to light treatments than that of phenolic acids [259,260].Water deficit and salt stress induce the synthesis of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA in hairy roots of S. miltiorrhiza [148,149,171,261].As mentioned above, PGRs also regulate the level of tanshinones and phenolic acids in S. miltiorrhiza.The interaction of JA signaling and light influence the synthesis of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by the SmHY5-SmBBX network [262].Ultraviolet-B and MeJA working together improves the level of cryptotanshinone, tanshinone I, tanshinone IIA in hairy roots of S. miltiorrhiza [158].As a result, the crosstalk between environmental stresses and PGRs are going to be the useful tool to increase the active compound production in S. miltiorrhiza.This is vital for the field cultivation and metabolic engineering of S. miltiorrhiza.

Conclusions and perspectives
In conclusion, considerable advancements have been achieved in the study of the roles of PGRs on the production of active compounds in S. miltiorrhiza, but much work remains to be done.With the enhancement of people's health awareness, there is a growing market need for S. miltiorrhiza.However, S. miltiorrhiza faces significant challenges, such as varying levels of germplasm quality, degradation of varieties, and replanting diseases, all of which can significantly affect its quality.Therefore, there is an urgent need to enhance the production of the active compounds in S. miltiorrhiza.Secondary metabolism in plants is influenced by both endogenous and exogenous stimuli.PGRs are important factors that affect the plant secondary metabolite accumulation.Currently, PGRs are primarily used in horticultural plants, and their associated regulatory mechanisms have been elucidated.However, the application and regulatory mechanisms of PGRs in medicinal plants remain poorly understood.Based on the above findings, various PGRs exhibit distinct regulatory mechanisms for the active compounds of S. miltiorrhiza.In addition, different active compounds exhibit the same or varying responses to different PGRs.JAs, ABA, GAs, SA, and KARs simultaneously regulate the level of phenolic acids and tanshinones.PAs induce phenolic acids biosynthesis.CKs may act as positive elicitors of phenolic acid and tanshinone accumulation.Accumulation of tanshinones may be promoted by AUXs, ET, and NO.Recently, it was found that SL may promote tanshinones accumulation.It is yet unknown how much each signaling pathway contributes in relation to these PGRs in S. miltiorrhiza.Although the regulatory effects of several PGRs, such as MeJA, ABA, SA, and GAs, on the active compound production in S. miltiorrhiza have been comprehensively studied, the effects of AUXs, CKs, ET, NO, PAs, KARs, and SLs, particularly on BRs and MT are not well documented.There are few reports on the crosstalk between PGRs in S. miltiorrhiza, especially regarding the post-transcriptional regulation and epigenetic regulatory effects of PGRs on the active compound production in S. miltiorrhiza.Moreover, CKs, BRs, GAs, ABA, and SLs share common precursors with tanshinone and may have a significant impact on tanshinone accumulation of S. miltiorrhiza.Crosstalk between these PGRs in S. miltiorrhiza should also be considered.Furthermore, during the growing season, environmental stressors frequently have an effect on the quality and yield of S. miltiorrhiza.MeJA, SA, ABA, ET, NO, and PAs are important in how plants response to environmental stimuli.However, the roles and signal transduction mechanisms of these PGRs in response to environmental stress in S. miltiorrhiza remain unclear.Up to now, the use of PGRs for S. miltiorrhiza cultivation is rare.The application of certain PGRs in S. miltiorrhiza cultivated fields may increase yield and decrease active compound production.For example, chlormequat chloride (CCC), an inhibitor of GA synthesis, is often used in the cultivated fields of S. miltiorrhiza to increase the yield in many production areas.However, the tanshinone content is likely to significantly drop as a result of this procedure.Hence, further studies are needed on the selection of appropriate PGRs and their application in terms of tissue, stage, concentration, frequency, and methods.Moreover, the effect of the crosstalk between PGRs and the surrounding environment on the active compound biosynthesis in S. miltiorrhiza requires further investigation.Ensuring the safety of medication is crucial, and the presence of PGR residues in medicinal materials is of great importance.The role and regulatory mechanisms of PGRs in the production of active compounds in S. miltiorrhiza have extensive potential for guiding S. miltiorrhiza cultivation and metabolic engineering of this plant species.

Fig. 8
Fig. 8 The relationship between PGRs and tanshinones and phenolic acids in S. miltiorrhiza.Dashed arrows denote multiple steps in the biosynthetic pathways.Solid arrows represent the regulatory function.Red lines represent the crosstalk of PGRs

Table 2
The effect of PGRs on the active compounds of S. miltiorrhiza