Functional Analysis of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Encoding Genes in Triterpene Saponin-Producing Ginseng    

Abstract

Ginsenosides are glycosylated triterpenes that are considered to be important pharmaceutically active components of the ginseng (Panax ginseng 'Meyer') plant, which is known as an adaptogenic herb. However, the regulatory mechanism underlying the biosynthesis of triterpene saponin through the mevalonate pathway in ginseng remains unclear. In this study, we characterized the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) concerning ginsenoside biosynthesis. Through analysis of full-length complementary DNA, two forms of ginseng HMGR (PgHMGR1 and PgHMGR2) were identified as showing high sequence identity. The steady-state mRNA expression patterns of PgHMGR1 and PgHMGR2 are relatively low in seed, leaf, stem, and flower, but stronger in the petiole of seedling and root. The transcripts of PgHMGR1 were relatively constant in 3-and 6-year-old ginseng roots. However, PgHMGR2 was increased five times in the 6-year-old ginseng roots compared with the 3-year-old ginseng roots, which indicates that HMGRs have constant and specific roles in the accumulation of ginsenosides in roots. Competitive inhibition of HMGR by mevinolin caused a significant reduction of total ginsenoside in ginseng adventitious roots. Moreover, continuous dark exposure for 2 to 3 d increased the total ginsenosides content in 3-year-old ginseng after the dark-induced activity of PgHMGR1. These results suggest that PgHMGR1 is associated with the dark-dependent promotion of ginsenoside biosynthesis. We also observed that the PgHMGR1 can complement Arabidopsis (Arabidopsis thaliana) hmgr1-1 and that the overexpression of PgHMGR1 enhanced the production of sterols and triterpenes in Arabidopsis and ginseng. Overall, this finding suggests that ginseng HMGRs play a regulatory role in triterpene ginsenoside biosynthesis.

Recent advances in steroidal saponins biosynthesis and in vitro production

“…Similarly in Medicago truncatula, silencing of Mkb1 (E3 ubiquitin ligase), that control the activity of HMGR results in accumulation of monoglycosylated saponins (Moses et al 2014a, b). This enzyme was characterized in different plants including Hevea brasiliensis (Chye et al 1991), potato (Bhattacharyya et al 1995), wheat (Aoyagi et al 1993), Camptotheca acuminate (Maldonado-Mendoza et al 1997), mulberry (Jain et al 2000), rice (Ha et al 2001), Capsicum annuum (Ha et al 2003), Ginkgo biloba (Shen et al 2006), Corylus avellana , Euphorbia pekinensi (Cao et al 2010), Salvia miltiorrhiza (Dai et al 2011), coffee (Tiski et al 2011), Glycyrrhiza uralensis (Rong et al 2011), Withania somnifera (Akhtar et al 2013), Dendrobium officinale (Zhang et al 2014a, b), Paris fargesii , Centella asiatica (Kalita et al 2015), Arabidopsis (Suzuki et al 2004), Cyanotis arachnoidea (Wang et al 2014a, b, c) and Panax ginseng (Kim et al 2014).…”

PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng

“…The HPLC separation was performed on a C18 column (5 µm, 4.6 × 250 mm) with water and acetonitrile as the mobile phase. The time and ratios of water and acetonitrile followed the protocol of Kim et al (2014). The flow rate of the mobile phase was 1.6 ml min -1 , and ginsenosides were monitored at a wavelength of 203 nm.…”

“…Ginsenosides were extracted according to the method described by Kim et al (2014). One gram aliquot of milled powder of freeze-dried roots was extracted twice for 1 h by refluxing with 80% methanol at 70 ºC.…”

“…Using the method from Kim et al (2014), freeze-dried plant material (rosette leaves, 200 mg) was powdered and then extracted twice with 1 ml CHCl 3 -MeOH (7:3) at room temperature. 5-α-cholestane (20 μg) was used as the internal standard.…”

Identification of a Highly Expressed 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene in the Root Tissue of <i>Taraxacum kok-saghyz</i>

“…Constitutive over expression of HMGR genes has proven to be effective in increasing production of selected isoprenoids and end-product sterols in plants [26]- [28]. The main organ of rubber synthesis and storage in Tk is the root.…”