Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins

Abstract

Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.

Transcription factor SmWRKY1 positively promote the biosynthesis of tanshinones inSalvia miltiorrhiza

“…A local transcription database of S. miltiorrhiza built up as reported previously (Shi et al, 2016b) was used for this research. One partial WRKY in high homology with other plants WRKYs while lack of 3’-terminal was chosen for further study.…”

“…MEGA 6 was applied to construct a phylogenetic tree by the neighbor-joining (NJ) method and 1000 replications were performed for bootstrap values. Multiple sequences alignment between SmWRKY1 and other plant WRKYs were carried out using Clustal X with default parameters (Shi et al, 2016b; Zhou et al, 2016a).…”

“…The constructed expression vector was transferred into Agrobacterium strain ASE and injected into forty-day-old tobacco leaves. GFP fluorescence was observed after 48h cultivation using the confocal microscope (Carl Zeiss) (Shi et al, 2016b; Zhou et al, 2016a).…”

“…The full-length coding sequence of SmWRKY1 with restriction sites Spe I and BstEII was cloned and inserted into modified pCAMBIA2300 sm vector (replace the small fragment digested by EcoR I and Hind III with the corresponding GFP-GUSA gene expression cassette from pCAMBIA1304 ) under the control of the CaMV 35S promoter to generate pCAMBIA2300 sm -SmWRKY1 as described before (Shi et al, 2016b). A. rhizogenes strain C58C1 containing pCAMBIA2300 sm -SmWRKY1 was used to infect the aseptic explants and the empty pCAMBIA2300 sm was regarded as the control.…”

“…A. rhizogenes strain C58C1 containing pCAMBIA2300 sm -SmWRKY1 was used to infect the aseptic explants and the empty pCAMBIA2300 sm was regarded as the control. The transformation procedure was the same as our previous study (Kai et al, 2011; Shi et al, 2014, 2016a, 2016b; Zhou et al, 2016a). Hairy roots in good state were sub-cultured and primer pairs CaMV35S-F23 and SmWRKY1-QR were used to identify the positive colony by polymerase chain reaction (PCR) analysis, meanwhile rolB gene in C58C1 was detected.…”