A novel WRKY34-bZIP3 module regulates phenolic acid and tanshinone biosynthesis in Salvia miltiorrhiza
Introduction
Salvia miltiorrhiza Bunge is a famous and highly-valued traditional Chinese medicine rich in phenolic acids and tanshinones, which have been widely used in curing cardiovascular and cerebrovascular diseases (Kai et al., 2011; Di et al., 2013; Xu and Song, 2017; Shi et al., 2016; Zhao et al., 2015; Ren et al., 2019). Phenolic acids are produced by the phenylpropane pathway and the tyrosine-derived pathway (Shi et al., 2019; Wang et al., 2015). Several gene members have been identified to participate in phenolic acid biosynthesis, namely phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), tyrosine aminotransferase (TAT), hydroxyphenylpyruvate reductase (HPPR), rosmarinic acid synthase (RAS) and cytochrome P450-dependent monooxygenase (CYP98A14) (Wang et al., 2015; Xiao et al., 2011; Huang et al., 2019; Liu et al., 2022). Tanshinones are synthesized from the common five-carbon precursor isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from mevalonate (MVA) present in the cytoplasm and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway locates in the plastid (Shi et al., 2016, 2021a, 2021b; Sun et al., 2019). Many genes have been identified to participate in tanshinone biosynthesis, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), geranylgeranyl diphosphate synthase (GGPPS), copalyl diphosphate synthase (CPS), kaurene synthase-like (KSL) and cytochrome P450-dependent monooxygenase (CYP76AH1) (Guo et al., 2013, 2016; Zhou et al., 2016). Besides, several CYP71Ds participated in the formation of the furan D-ring of transhinones (Ma et al., 2021). Due to the growth environment and artificial cultivation, high quality S. miltiorrhiza is in short supply. Therefore, it is of importance to improve the quality of S. miltiorrhiza.
Genetic engineering of key pathway genes has been testified as an effective method to elevate phenolic acid or tanshinone production which requires a thorough understanding of the biosynthetic pathway and its regulation mechanisms (Shi et al., 2014; Zhou et al., 2017; Fu et al., 2020). Besides, plant transcription factors (TFs) are involved in a variety of stress responses in plants, and can simultaneously regulate a variety of biosynthetic pathways (Suttipanta et al., 2011; Shi et al., 2021c). Many transcription factors (TFs) involved in the regulation of the biosynthesis of phenolic acids or tanshinones in S. miltiorrhiza have been identified. For example, SmERF128 can promote tanshinone biosynthesis by regulating MEP pathway genes such as SmCPS1, SmKSL1, and SmCYP76AH1 (Zhang et al., 2019). SmMYB9b-overexpression hairy roots produced higher content of tanshinones than that in control lines (Zhang et al., 2017). Also, phytohormone inducement is an alternative method to improve secondary metabolism (Liang et al., 2013; Yang et al., 2012). It was found that abscisic acid (ABA) elicitation is effective to improve the phenolic acid and tanshinone content in S. miltiorrhiza (Shi et al., 2021b), while there is limited understanding on ABA-responsive TFs participating in the simultaneous increase of phenolic acids and tanshinones (Deng et al., 2020; Shi et al., 2021a).
WRKY transcription factors are one of the plant-specific gene families, which contain a conserved domain consisting of 60 amino acids, of which the N-terminal is a conserved WRKYGQK motif, and C2HC (C-X7-C-X23-H-X1-C) or C2H2 (C-X4-5-C-X22-23-H-X1-H) (Rushton et al., 2010). According to the number of WRKY domains and zinc finger structures, the WRKY TFs can be divided into different subfamilies. WRKY TFs can specifically bind to W-box element (TTGACC/T) in the promoter region of target genes (Chen et al., 2017). WRKY TFs are not only involved in the regulation of biotic and abiotic stresses in plants, but also in other processes such as senescence, seed dormancy and development (Liu et al., 2016; Rushton et al., 2010; Robatzek and Somssich 2002), also in the secondary metabolism of plants (Suttipanta et al., 2011; Chen et al., 2017). For example, cotton GhWRKY25 increased differential tolerance of abiotic and biotic stress in transgenic tobacco (Liu et al., 2016). AaGSW1 increased the biosynthesis of artemisinin and dihydroartemisinin by binding to the W-box in the AaCYP71AV1 and AaORA promoters in Artemisia annua (Chen et al., 2017). SiWRKY8 promoted resistance to pathogen infection and mediated drought and salt stress tolerance in tomato (Gao et al., 2020). ABA-responsive Fortunella crassifolia WRKY40 enhanced transgenic tobacco and lemon more resistant to salt stress (Dai et al., 2018). Arabidopsis WRKY18, WRKY40 and WRKY60 are involved in plant sensitivity to ABA, salt and osmotic stress (Chen et al., 2010). Methyl jasmonate-inducible SmWRKY1 and SmWRKY2 positively regulated tanshinone biosynthesis in S. miltiorrhiza (Cao et al., 2018; Deng et al., 2019). However, ABA-responsive WRKY TFs playing a negative role in tanshinone or phenolic acid biosynthesis has not been reported.
The basic leucine zipper (bZIP) TF family is one of the major TFs participating in ABA signaling (Jakoby et al., 2002; Zhang et al., 2018). bZIP TFs play important roles in many aspects of plant processes such as development, pathogen resistance and secondary metabolism (Zhang et al., 2015, 2017; Deng et al., 2020). Arabidopsis thaliana bZIPfamily is comprised by 78 members, which can be grouped into 13 subfamilies. Notably, group A consisting of 13 members has the feature of direct binding to ABA-responsive element (ABRE: ACGTG-containing motif). Several group A members have been well studied. For instance, AabZIP1 activated AaADS and AaCYP71AV1 expression to promote artemisinin production A. annua (Zhang et al., 2015). ABA-responsive MdAREB2 promoted accumulation of soluble in apples by directly activating the expression of amylase genes and sugar transporter (Ma et al., 2017). ABF2, ABF3, and ABF4 promoted leaf senescence and degradation of chlorophyll in Arabidopsis (Gao et al., 2016). However, the role of ABA-responsive group A members involved in phenolic acid or tanshinone biosynthesis has been rarely reported.
In the present study, a novel ABA-responsive IIa WRKY TF at the transcriptional level by ABA hormone named SmWRKY34 was isolated from S. miltiorrhiza. Introduction of SmWRKY34 could reduce the production of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. On the contrary, knock out of SmWRKY34 obviously produced higher content of the two substances. Dual-LUC, Y1H and EMSA assays revealed that SmWRKY34 bind to W-box elements in the promoter regions of SmGGPPS and SmRAS. Besides, SmWRKY34 negatively regulated the transcript of a group A bZIP TF SmbZIP3, while SmbZIP3 played a positive role in phenolic acid and tanshinone accumulation by regulating SmTAT and two tanshinone-promoting TFs SmERF128 and SmMYB9b. In conclusion, our work presented a novel regulatory module of WRKY34-bZIP3 in ABA-promoted phenolic acid and tanshinone biosynthesis, providing new understandings into metabolic engineering of S. miltiorrhiza.
Section snippets
Plant materials and reagents
Aseptic S. miltiorrhiza plants were grown in the same condition as reported before (Kai et al., 2011; Shi et al., 2014). Seeds of N. benthamiana are sown and cultured for 4–5 weeks in pots supplemented with soil matrices (Zhou et al., 2016). All strains and plasmid vectors used in this study were kept in our laboratory. Prime STAR™ GXL DNA polymerase was purchased from Takara Biotechnology (China, Dalian) Co., Ltd. The RNA extraction kit, qRT-PCR kit, and reverse transcriptase were purchased
Isolation and characterization of SmWRKY34
A total of 61 WRKY members have been found from S. miltiorrhiza which could be sorted into three groups (I, II, III) and 8 subgroups (IC, IN, IIa, IIb, IIc, IId, IIe and III) (Li et al., 2015). Subgroup IIa WRKYs such as AtWRKY40 has been studied to play an important role in ABA signaling, and it has been testified that ABA was effective to promote phenolic acid or tanshinone accumulation (Chen et al., 2010; Shi et al., 2021a). AtWRKY40 was used as a probe to search in the ABA-induced
Discussion
The plant growth regulator ABA plays an important role in plant development and many physiological processes (Cutler et al., 2010). Previous studies have shown that exogenous ABA treatment could improve phenolic acid or tanshinone accumulation. For example, contents of tanshinone I, cryptotanshinone, dihydrotanshinone I and tanshinone IIA were significantly enhanced by treatment with 200 μM ABA treatment accompanied with upregulated expression of SmHMGR, SmDXR and SmDXS2 (Yang et al., 2012).
Authors' contributions
M.S., R.Z., and Y.Z. performed experiments. M.S. wrote the manuscript. S.Z., T.L., and L.W. helped analyzed the transcriptase data. K.L. helped HPLC analysis. M.S., S.L., Y.W., W.Z., and Q.H. revised the manuscript. G.K. designed the experiments and conceived the project, provided overall supervision of the study.
Author statement
The authors declare no financial or commercial conflict of interest.
Declaration of competing interest
The authors declare that they have no competing interests.
Acknowledgements
This work was supported by National Natural Science Fund of China [82073963, 31571735, 81522049]; The Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province [2021C02074]; Zhejiang Provincial Ten Thousand Program for Leading Talents of Science and Technology Innovation [2018R52050]; Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents; Zhejiang Chinese Medical University Research Foundation [2021JKZDZC06]; Opening
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