Functional pleiotropism, diversity, and redundancy of Salvia miltiorrhiza Bunge JAZ family proteins in jasmonate-induced tanshinone and phenolic acid biosynthesis

Abstract

Jasmonate (JA) signaling regulates plant growth and development, biotic and abiotic stress tolerance, and primary and secondary metabolism biosynthesis. It is extensively modulated by JA-ZIM-domain (JAZ) family genes. In previous work, we obtained nine SmJAZ genes of Salvia miltiorrhiza and proved that SmJAZ8 was the core repressor of JA-induced tanshinone and phenolic acid biosynthesis. Here, we demonstrate that SmJAZ3 and SmJAZ4 act as repressors of JA-induced biosynthesis of tanshinones and salvianolic acid B (Sal B). This suggests that SmJAZ3/4 are functionally redundant in tanshinone and Sal B biosynthesis. SmJAZ1/2/5/6/9 are activators of JA-induced tanshinone biosynthesis and repressors of JA-induced Sal B biosynthesis. This demonstrates the redundancy and diversity of SmJAZ1/2/5/6/9 functions. Besides, SmJAZ10 inhibited JA-induced Sal B synthesis, but had no effect on the synthesis of tanshinone. Two-hybrid screening (Y2H) showed that SmJAZs formed homologous or heterogeneous dimers. Y2H and firefly luciferase complementation imaging (LCI) assays revealed that SmJAZs also formed a complex regulatory network with SmMYC2a, SmMYC2b, SmMYB39, and SmPAP1. Quantitative reverse transcription-PCR (qRT-PCR) indicated that SmJAZs regulated each other at the transcriptional level. Herein, we prove that SmJAZs have functional pleiotropism, diversity, and redundancy in JA-induced tanshinone and phenolic acid biosynthesis. This study provides an important clue for further understanding the inherent biological significance and molecular mechanisms of the JAZ family as the gene number increases during plant evolution.

References

[1] Ma P, Liu J, Zhang C et al. Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge. Appl Biochem Biotechnol. 2013; 170: 1253-62.

[2] Ma P, Liu J, Osbourn A et al. Regulation and metabolic engineering of tanshinone biosynthesis. RSC Adv. 2015; 5: 18137-44.

[3] Di P, Zhang L, Chen J et al. 13C Tracer reveals phenolic acids biosynthesis in hairy root cultures of Salvia miltiorrhiza . ACS Chem Biol. 2013; 8: 1537-48.

[4] Huot B, Yao J, Montgomery BL et al. Growth-defense tradeoffs in plants: a balancing act to optimize fitness. Mol Plant. 2014; 7: 1267-87.

[5] Farmer EE . Plant biology: jasmonate perception machines. Nature. 2007; 448: 659-60.

[6] Zhang J, Zhou L, Zheng X et al. Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots. Plant Cell Rep. 2017; 36: 1297-309.

[7] Li L, Wang D, Zhou L et al. JA-responsive transcription factor SmMYB97 promotes phenolic acid and tanshinone accumulation in Salvia miltiorrhiza . J Agric Food Chem. 2020; 68: 14850-62.

[8] Zhou Y, Sun W, Chen J et al. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza . Sci Rep. 2016; 6: 22852.

[9] Zheng H, Jing L, Jiang X et al. The ERF-VII transcription factor SmERF73 coordinately regulates tanshinone biosynthesis in response to stress elicitors in Salvia miltiorrhiza . New Phytol. 2021; 231: 1940-55.

[10] Sun M, Shi M, Wang Y et al. The biosynthesis of phenolic acids is positively regulated by the JA-responsive transcription factor ERF115 in Salvia miltiorrhiza . J Exp Bot. 2019; 70: 243-54.

[11] Zhou W, Shi M, Deng C et al. The methyl jasmonate-responsive transcription factor SmMYB1 promotes phenolic acid biosynthesis in Salvia miltiorrhiza . Hortic Res. 2021; 8: 10.

[12] Hao G, Jiang X, Feng L et al. Cloning, molecular characterization and functional analysis of a putative R2R3-MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba . Plant Cell Tissue Organ Cult. 2016; 124: 151-68.

[13] Du T, Niu J, Su J et al. SmbHLH37 functions antagonistically with SmMYC2 in regulating jasmonate-mediated biosynthesis of phenolic acids in Salvia miltiorrhiza . Front Plant Sci. 2018; 9: 1720.

[14] Huang Q, Sun M, Yuan T et al. The AP2/ERF transcription factor SmERF1L1 regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza . Food Chem. 2019; 274: 368-75.

[15] Lu XY, Liang XY, Li X et al. Genome-wide characterisation and expression profiling of the LBD family in Salvia miltiorrhiza reveals the function of LBD50 in jasmonate signaling and phenolic biosynthesis. Ind Crop Prod. 2020; 144: 112006.

[16] Han GZ . Evolution of jasmonate biosynthesis and signaling mechanisms. J Exp Bot. 2017; 68: 1323-31.

[17] Garrido-Bigotes A, Valenzuela-Riffo F, Figueroa CR . Evolutionary analysis of JAZ proteins in plants: an approach in search of the ancestral sequence. Int J Mol Sci. 2019; 20: 5060.

[18] Shen J, Zou Z, Xing H et al. Genome-wide analysis reveals stress and hormone responsive patterns of JAZ family genes in Camellia sinensis . Int J Mol Sci. 2020; 21: 2433.

[19] Zheng Y, Chen X, Wang P et al. Genome-wide and expression pattern analysis of JAZ family involved in stress responses and postharvest processing treatments in Camellia sinensis . Sci Rep. 2020; 10: 2792.

[20] Wang P, Yu S, Han X et al. Identification, molecular characterization and expression of JAZ genes in Lycoris aurea . PLoS One. 2020; 15: e0230177.

[21] Han Y, Luthe D . Identification and evolution analysis of the JAZ gene family in maize. BMC Genomics. 2021; 22: 256.

[22] Monte I, Franco-Zorrilla JM, García-Casado G et al. A single JAZ repressor controls the jasmonate pathway in Marchantia polymorpha . Mol Plant. 2019; 12: 185-98.

[23] Chini A, Fonseca S, Fernández G et al. The JAZ family of repressors is the missing link in jasmonate signalling. Nature. 2007; 448: 666-71.

[24] Yan Y, Stolz S, Chételat A et al. A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell. 2007; 19: 2470-83.

[25] Chung HS, Cooke TF, DePew CL et al. Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling. Plant J. 2010; 63: 613-22.

[26] Demianski AJ, Chung KM, Kunkel BN . Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis . Mol Plant Pathol. 2012; 13: 46-57.

[27] Yang DL, Yao J, Mei CS et al. Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proc Natl Acad Sci USA. 2012; 109: E1192-200.

[28] Pauwels L, Ritter A, Goossens J et al. The RING E3 ligase KEEP ON GOING modulates JASMONATE ZIM-DOMAIN12 stability. Plant Physiol. 2015; 169: 1405-17.

[29] Thireault C, Shyu C, Yoshida Y et al. Repression of jasmonate signaling by a non-TIFY JAZ protein in Arabidopsis . Plant J. 2015; 82: 669-79.

[30] Thines B, Katsir L, Melotto M et al. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. Nature. 2007; 448: 661-5.

[31] Chung HS, Koo AJK, Gao X et al. Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory. Plant Physiol. 2008; 146: 952-64.

[32] Ramirez V, Coego A, Lopez A et al. Drought tolerance in Arabidopsis is controlled by the OCP3 disease resistance regulator. Plant J. 2009; 58: 578-91.

[33] Jiang Y, Liang G, Yang S et al. Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid- and auxin-mediated signaling in jasmonic acid-induced leaf senescence . Plant Cell. 2014; 26: 230-45.

[34] Hu Y, Jiang L, Wang F et al. Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis . Plant Cell. 2013; 25: 2907-24.

[35] Oblessuc PR, Obulareddy N, DeMott L et al. JAZ4 is involved in plant defense, growth, and development in Arabidopsis . Plant J. 2020; 101: 371-83.

[36] Sehr EM, Agusti J, Lehner R et al. Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation. Plant J. 2010; 63: 811-22.

[37] Yan H, Yoo MJ, Koh J et al. Molecular reprogramming of Arabidopsis in response to perturbation of jasmonate signaling. J Proteome Res. 2014; 13: 5751-66.

[38] Meng L, Zhang T, Geng S et al. Comparative proteomics and metabolomics of JAZ7-mediated drought tolerance in Arabidopsis . J Proteome. 2019; 196: 81-91.

[39] Han X, Zhang M, Yang M et al. Arabidopsis JAZ proteins interact with and suppress RHD6 transcription factor to regulate jasmonate-stimulated root hair development . Plant Cell. 2020; 32: 1049-62.

[40] Chung HS, Howe GA . A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis . Plant Cell. 2009; 21: 131-45.

[41] Shyu C, Figueroa P, DePew CL et al. JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis . Plant Cell. 2012; 24: 536-50.

[42] de Torres Zabala M, Zhai B, Jayaraman S et al. Novel JAZ cooperativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection . New Phytol. 2016; 209: 1120-34.

[43] Shi M, Zhou W, Zhang J et al. Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins. Sci Rep. 2016; 6: 20919.

[44] Ge Q, Zhang Y, Hua WP et al. Combination of transcriptomic and metabolomic analyses reveals a JAZ repressor in the jasmonate signaling pathway of Salvia miltiorrhiza . Sci Rep. 2015; 5: 14048.

[45] Zhou M, Memelink J . Jasmonate-responsive transcription factors regulating plant secondary metabolism. Biotechnol Adv. 2016; 34: 441-9.

[46] Yang D, Ma P, Liang X et al. Metabolic profiles and cDNA-AFLP analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib. PLoS One. 2012; 7: e29678.

[47] Pei T, Ma P, Ding K et al. SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J Exp Bot. 2018; 69: 1663-78.

[48] Wu H, Ye H, Yao R et al. OsJAZ9 acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice. Plant Sci. 2015; 232: 1-12.

[49] Fu J, Wu H, Ma S et al. OsJAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice. Front Plant Sci. 2017; 8: 2108.

[50] Yamada S, Kano A, Tamaoki D et al. Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant Cell Physiol. 2012; 53: 2060-72.

[51] Taniguchi S, Hosokawa-Shinonaga Y, Tamaoki D et al. Jasmonate induction of the monoterpene linalool confers resistance to rice bacterial blight and its biosynthesis is regulated by JAZ protein in rice. Plant Cell Environ. 2014; 37: 451-61.

[52] Oh Y, Baldwin IT, Galis I . NaJAZh regulates a subset of defense responses against herbivores and spontaneous leaf necrosis in Nicotiana attenuata plants. Plant Physiol. 2012; 159: 769-88.

[53] Oh Y, Baldwin IT, Galis I . A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants. PLoS One. 2013; 8: e57868.

[54] Shoji T, Ogawa T, Hashimoto T . Jasmonate-induced nicotine formation in tobacco is mediated by tobacco COI1 and JAZ genes. Plant Cell Physiol. 2008; 49: 1003-12.

[55] Hu H, He X, Tu L et al. GhJAZ2 negatively regulates cotton fiber initiation by interacting with the R2R3-MYB transcription factor GhMYB25-like. Plant J. 2016; 88: 921-35.

[56] Sun H, Chen L, Li J et al. The JASMONATE ZIM-domain gene family mediates JA signaling and stress response in cotton. Plant Cell Physiol. 2017; 58: 2139-54.

[57] He X, Zhu L, Wassan GM et al. GhJAZ2 attenuates cotton resistance to biotic stresses via the inhibition of the transcriptional activity of GhbHLH171. Mol Plant Pathol. 2018; 19: 896-908.

[58] Xia XC, Hu QQ, Li W et al. Cotton (Gossypium hirsutum) JAZ3 and SLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation . Plant Cell Tissue Org Cult. 2018; 133: 249-62.

[59] Xiao Y, Gao S, di P et al. Lithospermic acid B is more responsive to silver ions (Ag+) than rosmarinic acid in Salvia miltiorrhiza hairy root cultures. Biosci Rep. 2009; 30: 33-40.

[60] Yu J, Zhang Y, Di C et al. JAZ7 negatively regulates dark-induced leaf senescence in Arabidopsis . J Exp Bot. 2016; 67: 751-62.

[61] Chini A, Fonseca S, Chico JM et al. The ZIM domain mediates homo- and heteromeric interactions between Arabidopsis JAZ proteins. Plant J. 2009; 59: 77-87.

[62] Pandey SP, Roccaro M, Schon M et al. Transcriptional reprogramming regulated by WRKY18 and WRKY40 facilitates powdery mildew infection of Arabidopsis . Plant J. 2010; 64: 912-23.

[63] Wu F, Deng L, Zhai Q et al. Mediator subunit MED25 couples alternative splicing of JAZ genes with fine-tuning of jasmonate signaling. Plant Cell. 2020; 32: 429-48.

[64] Seo JS, Joo J, Kim MJ et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J. 2011; 65: 907-21.

[65] Yang YP, Guo J, Yan P et al. Transcriptome profiling identified multiple jasmonate ZIM-domain proteins involved in the regulation of alkaloid biosynthesis in tobacco BY-2 cells. Plant Mol Biol Rep. 2015; 33: 153-66.

[66] Kazan K, Manners JM . MYC2: the master in action. Mol Plant. 2013; 6: 686-703.

[67] Zhou Y, Sun W, Chen J et al. Author correction: SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza . Sci Rep. 2020; 10: 7201.

[68] Pauwels L, Goossens A . The JAZ proteins: a crucial interface in the jasmonate signaling cascade. Plant Cell. 2011; 23: 3089-100.

[69] Zhou W, Li S, Maoz I et al. SmJRB1 positively regulates the accumulation of phenolic acid in salvia miltiorrhiza. Ind Crop Prod. 2021; 164: 113417.

[70] Eddy SR . Profile hidden Markov models. Bioinformatics. 1998; 14: 755-63.

[71] Kumar S, Stecher G, Li M et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-9.

[72] Yang YF, Hou S, Cui G et al. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Salvia miltiorrhiza . Mol Biol Rep. 2010; 37: 507-13.

[73] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001; 25: 402-8.

[74] Chen H, Zou Y, Shang Y et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 2008; 146: 368-76.