Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors

Abstract

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species.

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes

“…In both cases, the MJ-induced accumulation in abietane diterpenes was higher than that triggered by overexpressing individually the plant and the bacterial DXS or DXR genes, corroborating the notion that simultaneous overexpression of multiple biosynthetic genes is often necessary to efficiently increase the accumulation of valuable plant secondary metabolites. It can be envisaged that simultaneous overexpression of the bacterial DXR gene in combination with other biosynthetic genes encoding limiting enzymatic steps of this metabolic pathway, identified in our previous studies [21,22], might positively further affect the accumulation of abietane diterpenes in S. sclarea hairy roots.…”

“…(e.g., Salvia aethiopis and others) have been shown to be cytotoxic against different tumor lines [4][5][6][7][8], but their low amount has hampered the possibility of accurately investigating the underlying mechanisms of its antiproliferative activity. In an effort to understand the contribution of different biosynthetic genes to a higher accumulation of bioactive abietane diterpenes in S. sclarea hairy roots, we targeted this metabolic pathway by different and complementary approaches, including the transcriptional reprograming of multiple genes by MJ elicitation [21] or by overexpressing MYC and WRKY transcription factors [22]. From both studies, it was clearly evident that the expression level of the DXS and DXR genes were highly correlated with an enhanced content of abietane diterpenes in S. sclarea hairy roots, confirming our previous data obtained by overexpressing the Arabidopsis thaliana DXS1 and DXR genes [8].…”

“…We have also demonstrated that S. sclarea hairy roots overexpressing the Arabidopsis DXS and DXR genes produced a 2-fold and about 4-fold higher content of abietane-type diterpenes, respectively, especially aethiopinone [8]. In addition, the expression of the genes encoding these two enzymes acting upstream of the MEP pathway is highly correlated to an enhanced accumulation of abietane diterpenes in S. sclarea hairy roots elicited with MJ or coronatine [21] as well as in hairy roots overexpressing the WRKY or MYC2 transcription factor [22]. In trying to further optimize the conditions to produce an increased content of aethiopinone from S. sclarea hairy roots, cyanobacterial orthologous genes encoding these two first enzymes of the MEPderived isoprenoid pathway were ectopically expressed in S. scla- rea hairy roots.…”

WRKY transcription factors: evolution, binding, and action

“…Jasmonate-responsive TFs, such as WRKYs and MYCs, are involved in the regulation of plant metabolism. The ectopic expression of AtWRKY18 and AtWRK40 together with AtMYC2 activated the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway in Salvia sclarea hairy roots and consequently elevated the abietane diterpene content (Alfieri et al 2018). Catharanthus roseus produces various terpenoid indole alkaloids that are of pharmaceutical importance.…”

Transcriptional Factors Regulate Plant Stress Responses Through Mediating Secondary Metabolism

“…Production of abietane-type diterpenoids was studied in Salvia sclarea to be regulated by SsWRKY18, SsWRKY40, and SsMYC2. The overexpression lines of these TFs significantly accumulated higher abietane-type diterpenoids that were responsible for resistance against various bacterial and fungal species [73]. In addition, the silencing of WsWRKY1 in Withania somnifera decreased phytosterol accumulation and consequently increased the susceptibility to bacteria, fungi, and insect infestation [71].…”