Proteomic analysis reveals novel insights into tanshinones biosynthesis in Salvia miltiorrhiza hairy roots

Angela Contreras,Ruddy Wattiez,Baptiste Leroy,Pierre-Antoine Mariage

doi:10.1038/s41598-019-42164-3

Abstract

Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.

Highlights

Salvia miltiorrhiza Bunge, commonly called danshen, is widely used in traditional Chinese medicine, to treat cardiovascular diseases and inflammatory processes[1,2,3]
Our results confirmed the combination of biotic elicitor (YE) with abiotic elicitor (Ag+) is more effective to produce tanshinones than a single elicitor; observations previously reported in S. miltiorrhiza hairy roots by Wang et al.[9] and other goups[8,42]
This concentration was comparable to that reported by Cheng et al.[42] using 2.5 g L−1 yeast extract (YE) + 100 μM Ag+ after 5 days-elicitation, but much more higher than those reached by Wang et al.[12] after 5 days of elicitation using 100 g L−1 YE and 3 mM Ag+ (0.7 mg g−1 dry weight (DW)), Kai et al.[8] after 9 days using 100 mg L−1 YE and 0.03 mM Ag+ (2.08 mg/g DW), Ge et al.[23] after 8 days using 30 μM Ag+ (0.96 mg g−1 DW) and 0.1 g L−1 YE (2.48 mg g−1 DW)

Summary

Introduction

Salvia miltiorrhiza Bunge, commonly called danshen, is widely used in traditional Chinese medicine, to treat cardiovascular diseases and inflammatory processes[1,2,3]. Many pharmacological active compounds are naturally produced in response to different kind of environmental stresses as temperature changes, drought, pathogens attack and osmotic stress[3,16,17,18,19], their production in tissue cultures could be stimulated by biotic and abiotic agents through a strategy called elicitation[20] Elicitors such as methyl jasmonate (MeJA), yeast extract (YE), salicylic acid (SA) and AgNO3 (Ag+), used individually or in combination, can efficiently stimulate tanshinone accumulation in S. miltiorrhiza hairy roots[9,21,22]. Only one such study has been reported by Wang et al.[12] following the addition of YE and Ag+ to S. miltiorrhiza hairy roots during the first 5 days Under this elicitation 64 proteins were identified showing activation of Ca2+/calmodulin signaling pathway, burst of reactive oxygen species and enhancing energy and carbon metabolism. Use of this technique may significantly improve detectability of low abundant proteins such as transcription factors, kinases, and transport proteins among others

Methods

Results

Conclusion