Citation: Szymczyk, P.; Ku´ zma, L.; Jele ´ n, A.; Balcerczak, E.; Majewska, M. Isolation of Salvia miltiorrhiza Kaurene Synthase-like (KSL) Gene Promoter and Its Regulation by Ethephon and Yeast Extract. Genes 2023, 14, 54. https://doi.org/10.3390/ genes14010054 Academic Editor: Qinghu Ma Received: 24 November 2022 Revised: 19 December 2022 Accepted: 21 December 2022 Published: 24 December 2022 Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article Isolation of Salvia miltiorrhiza Kaurene Synthase-like (KSL) Gene Promoter and Its Regulation by Ethephon and Yeast Extract Piotr Szymczyk 1, * , Lukasz Ku ´ zma 1 , Agnieszka Jele ´ n 2 , Ewa Balcerczak 2 and Malgorzata Majewska 1 1 Department of Biology and Pharmaceutical Botany, Medical University of Lód´ z, Muszy ´ nskiego 1, 90-151 Lód´ z, Poland 2 Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lód´ z, Muszy ´ nskiego 1, 90-151 Lód´ z, Poland * Correspondence: piotr.szymczyk@umed.lodz.pl Abstract: The presented study describes the regulation of the promoter region of the Salvia miltiorrhiza kaurene synthase-like gene (SmKSL) by ethylene and yeast extract. The isolated fragment is 897 bp and is composed of a promoter (763 bp), 5 0 UTR (109 bp), and a short CDS (25 bp). The initial in silico analysis revealed the presence of numerous putative cis-active sites for trans-factors responding to different stress conditions. However, this study examines the influence of ethylene and yeast extract on SmKSL gene expression and tanshinone biosynthesis regulation. The results of 72h RT-PCR indicate an antagonistic interaction between ethylene, provided as ethephon (0.05, 0.10, 0.25, and 0.50 mM), and yeast extract (0.5%) on SmKSL gene expression in callus cultures of S. miltiorrhiza. A similar antagonistic effect was observed on total tanshinone concentration for up to 60 days. Ethylene provided as ethephon (0.05, 0.10, 0.25, and 0.50 mM) is a weak inducer of total tanshinone biosynthesis, increasing them only up to the maximum value of 0.67 ± 0.04 mg g -1 DW (60-day induction with 0.50 mM ethephon). Among the tanshinones elicited by ethephon, cryptotanshinone (52.21%) dominates, followed by dihydrotanshinone (45.00%) and tanshinone IIA (3.79%). In contrast, the 0.5% yeast extract strongly increases the total tanshinone concentration up to a maximum value of 13.30 ± 1.09 mg g -1 DW, observed after 50 days of induction. Yeast extract and ethylene appear to activate different fragments of the tanshinone biosynthesis route; hence the primary tanshinones induced by yeast extract were cryptotanshinone (81.42%), followed by dihydrotanshinone (17.06%) and tanshinone IIA (1.52%). Keywords: callus culture; promoter; cis-active element; tanshinone; ethylene; yeast extract 1. Introduction S. miltiorrhiza Bunge holds a distinguished position among other medicinal plants since it is a traditional source of medically-active tanshinones. The medicinal properties of tanshinones have been exploited in China since ancient times to treat chronic heart failure [1]. The clinical applications of S. miltiorrhiza have recently broadened to include neuropathic pain, hyperlipidemia, alcoholism, Parkinson’s, and Alzheimer’s disease [2–4]. Its popularity in research can be attributed to its relatively short life cycle, ease of prop- agation, and availability of genome transformation systems. In addition, it has a range of well-developed in vitro culture systems and a relatively small draft genome sequence of about 538 MB, containing 30,478 predicted genes [5]. Tanshinones are predominantly acquired from the roots of S. miltiorrhiza field plants. However, this source is becoming steadily impoverished by climate change, soil pollution, water deficit, and incremental stepping of arable area, making traditional production more difficult [6]. The most efficient alternative sources of S. miltiorrhiza plant material are hairy roots and calli grown on solid medium or as suspension callus cell cultures [7–9]. These cultures provide a continuous, Genes 2023, 14, 54. https://doi.org/10.3390/genes14010054 https://www.mdpi.com/journal/genes