Accumulation of Polyphenols and Associated Gene Expression in Hairy Roots of Salvia viridis Exposed to Methyl Jasmonate.

Methyl jasmonate (MJA), a signaling molecule in stress pathways, can be used to induce secondary metabolite synthesis in plants. The present study examines its effects on the growth of Salvia viridis hairy roots, and the accumulation of bioactive compounds, and correlates it with the expression of genes involved in the phenylpropanoid pathway. To our knowledge, this study represents the first exploration of elicitation in S. viridis culture and the first comprehensive analysis of MJA's influence on such a wide array of genes within the polyphenol metabolic pathway in the Salvia genus. Plants were treated with 50 and 100 µM MJA, and samples were collected at intervals of one, three, five, and seven days post-elicitation. HPLC analysis revealed that MJA stimulated the accumulation of all tested compounds, with a 30% increase (38.65 mg/g dry weight) in total polyphenol content (TPC) on day five. Quantitative real-time polymerase chain reaction (RT-PCR) analysis demonstrated a significant increase in the expression of the phenylpropanoid pathway genes-TAT (tyrosine aminotransferase), HPPR (4-hydroxyphenylpyruvate reductase), PAL (phenylalanine ammonia-lyase), C4H (cinnamic acid 4-hydroxylase), 4CL (4-coumarate-CoA ligase), and RAS (rosmarinic acid synthase)-following MJA treatment. For the majority of the genes, this increase was observed after the first day of treatment. Importantly, our present results confirm strong correlations of the analyzed gene expression with polyphenol biosynthesis. These findings support the notion that hairy roots provide a promising biotechnological framework for augmenting polyphenol production. Additionally, the combination of elicitor treatment and transgenic technology emerges as a viable strategy to enhance the biosynthesis of these valuable metabolites.

The Expression Profiles of the Salvia miltiorrhiza 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase 4 Gene and Its Influence on the Biosynthesis of Tanshinones

Salvia miltiorrhiza is a medicinal plant that synthesises biologically-active tanshinones with numerous therapeutic properties. An important rate-limiting enzyme in the biosynthesis of their precursors is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). This study presents the organ-specific expression profile of the S. miltiorrhiza HMGR4 gene and its sensitivity to potential regulators, viz. gibberellic acid (GA3), indole-3-acetic acid (IAA) and salicylic acid (SA). In addition, it demonstrates the importance of the HMGR4 gene, the hormone used, the plant organ, and the culture environment for the biosynthesis of tanshinones. HMGR4 overexpression was found to significantly boost the accumulation of dihydrotanshinone I (DHTI), cryptotanshinone (CT), tanshinone I (TI) and tanshinone IIA (TIIA) in roots by 0.44 to 5.39 mg/g dry weight (DW), as well as TIIA in stems and leaves. S. miltiorrhiza roots cultivated in soil demonstrated higher concentrations of the examined metabolites than those grown in vitro. GA3 caused a considerable increase in the quantity of CT (by 794.2 µg/g DW) and TIIA (by 88.1 µg/g DW) in roots. In turn, IAA significantly inhibited the biosynthesis of the studied tanshinones in root material.

Establishment of hairy root cultures of Salvia bulleyana Diels for production of polyphenolic compounds.

This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.