Genome-Wide Identification of the TIFY Family in Salvia miltiorrhiza Reveals That SmJAZ3 Interacts With SmWD40-170, a Relevant Protein That Modulates Secondary Metabolism and Development

Abstract

Salvia miltiorrhiza Bunge (S. miltiorrhiza), a traditional Chinese medicinal herb, contains numerous bioactive components with broad range of pharmacological properties. By increasing the levels of endogenous jasmonate (JA) in plants or treating them with methyl jasmonate (MeJA), the level of tanshinones and salvianolic acids can be greatly enhanced. The jasmonate ZIM (JAZ) proteins belong to the TIFY family, and act as repressors, releasing targeted transcriptional factors in the JA signaling pathway. Herein, we identified and characterized 15 TIFY proteins present in S. miltiorrhiza. Quantitative reverse transcription PCR analysis indicated that the JAZ genes were all constitutively expressed in different tissues and were induced by MeJA treatments. SmJAZ3, which negatively regulates the tanshinones biosynthesis pathway in S. miltiorrhiza and the detailed molecular mechanism is poorly understood. SmJAZ3 acts as a bait protein to capture and identify a WD-repeat containing the protein SmWD40-170. Further molecular and genetic analysis revealed that SmWD40-170 is a positive regulator, promoting the accumulation of secondary metabolites in S. miltiorrhiza. Our study systematically analyzed the TIFY family and speculated a module of the JAZ-WD40 complex provides new insights into the mechanisms regulating the biosynthesis of secondary metabolites in S. miltiorrhiza.

Keywords: Salvia miltiorrhiza; SmJAZ3; SmWD40-170; TIFY proteins; jasmonate.

FIGURE 1

Neighbor-joining phylogenetic tree of TIFY proteins from Salvia miltiorrhiza, Arabidopsis, Vitis vinifera, and Oryza sativa. The tree was developed using MEGA 6 software and the bootstrap method (1000 replicates).

FIGURE 2

(A) Distribution of conserved domains within SmTIFY, SmJAZ, SmPPD, and SmZML proteins. Relative positions of domains within each protein are shown in different colors. (B) Gene structures of SmTIFY gene family. Exon, yellow-filled boxes; intron, black single lines. (C) Sequence logos of TIFY domains from SmTIFY proteins. (D) Sequence logos of Jas domains from SmTIFY proteins.

FIGURE 3

Relative expression of SmTIFY genes in root, stem, leave, and flower. All data represent averages of three biological replicates, error bars indicate SD. Statistical significance was determined using the Student’s t-test (*p < 0.05, **p < 0.01) between root, stem, leaf, and flower.

FIGURE 4

Relative expression level of SmTIFY genes in S. miltiorrhiza plants treated with mock control and 100 μM MeJA. All data represent averages of three biological replicates, error bars indicate SD. Statistical significance was determined by the Student’s t-test (*p < 0.05, **p < 0.01).

FIGURE 5

SmJAZ3 interacts with SmWD40-170. (A) Schematic diagrams show domain constructs of SmJAZ3. (B) Y2H assays was used to test the interactions of SmWD40-170 with different domains of SmJAZ3. (C) BiFC assays was used to detect the interaction between SmJAZ3 and SmWD40-170.

FIGURE 6

Secondary metabolites contents in control and SmWD40-170 transgenic roots of S. miltiorrhiza. Comparisons of total phenolic acid, total flavonoids, rosmarinic acid, salvianolic acid B, tanshinone IIA, and cryptotanshinone concentration among transgenic and control lines. All data represent averages of three biological replicates, error bars indicate SD. Statistical significance was determined using the Student’s t-test (**p < 0.01).

FIGURE 7

Expression analysis of salvianolic acids and tanshinones biosynthesis genes. RT-qPCR analyses of the key enzyme genes of salvianolic acids biosynthetic pathway in OE (A) and RNAi (C) lines. RT-qPCR analyses of the key enzyme genes of tanshinones biosynthetic pathway in OE (B) and RNAi (D) lines. All data represent averages of three biological replicates, error bars indicate SD. Statistical significance was determined using the Student’s t-test (*p < 0.05, **p < 0.01). PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, hydroxycinnamate-CoA ligase; TAT, tyrosine aminotransferase; HPPR, hydroxyl phenylpyruvate reductase; RAS, rosmarinic acid synthase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; CPS, copalyl diphosphate synthase; KSL, kaurene synthase-like synthase.

FIGURE 8

Morphological differences in the control and SmWD40-170 transgenic lines. (A/B/C,D/E/F,G/H/I,J/K/L) represent three independent repeats. (A–F) Control and transgenic seedlings cultured in soil for 2 months. (G–I) Control and transgenic roots. (J–L) Control and transgenic leaves. (M) Comparison of the root length in control and transgenic lines. (N) Comparison of the leaf size in control and transgenic lines. All data represent averages of three biological replicates, error bars indicate SD. Statistical significance was determined using the Student’s t-test (*p < 0.05, **p < 0.01).

FIGURE 9

Proposed module of the roles of JA in regulating secondary metabolites biosynthesis as well as growth and development. When treating with exogenous JA, the complex formation between the JA-Ile and COI1 promotes SmJAZ3 degradation via the 26S proteasome, and release the positive regulators such as WD40, then enhances the activities of enzymes to promote secondary metabolites biosynthesis. In addition, SmWD40-170 protein may regulate growth and development through JA-dependent or JA-independent pathway. Arrows, positive regulation; blunt ends, negative regulation; dotted line, uncertified process.