Full Text - Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression

Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression

¹Wuhan University, College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan, PR China

Financial support: This work was supported by the National Natural Science Foundation of China (30370152) and the Chinese 111 project (B06018).

Background: The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome. Although the stimulation of diosgenin accumulation by ethylene has been reported in a few of plant species, its regulation is not yet characterized at the molecular level, the underlying molecular mechanism remains elusive.

Results: In this study, the effects of ethylene on diosgenin biosynthesis in in vitro cultures of D. zingiberensis were described. The results showed that, in samples treated with ethylene at concentration E3 (10⁴ dilution of 40% ethephon), the diosgenin biosynthesis was significantly promoted in comparison with the control samples. Treatment with high concentrations of ethylene had inhibitory effect, whereas with low concentration of the gas elicitor brought about no detectable deleterious effect on the growth rate and diosgenin content of the cultures. The considerable increase of diosgenin level in in vitro cultured Dioscorea zingiberensis by ethylene application is accompanied by the concomitant increase of soluble proteins and chlorophyll content. The gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase but not of squalene synthase or farnesyl pyrophosphate synthase were up-regulated by applied ethylene.

Conclusions: Our results suggest that ethylene treatment enhanced diosgenin accumulation via up-regulation of the gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Diosgenin, (25R)-Spirost-5-en-3β-ol (Figure 1), is a steroidal sapogenin isolated from the plants (Marker et al. 1940). It is one of the most important raw materials for steroid drugs manufacturing. Diosgenin represents the main starting material for commercial synthesis of sex drugs and corticosteroids such as cortisone, testosterone, and progesterone. Estrogenic, progesterogenic and anti-inflammatory effects have been hypothesized for diosgenin due to its structural similarity to estrogen and progesterone precursors (Dewick, 2002). Anticancer activity, controlling hyperlipidemia, inhibiting melanogenesis, delaying skin aging at the time of climacteric have also been demonstrated (Lee et al. 2007; Tada et al. 2009; Yan et al. 2009; Gong et al. 2010). The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome (Huai et al. 1989; Ding et al. 1991; Cheng et al. 2008).

In general, the amount of diosgenin naturally accumulated in plant tissues is small, but can be increased by various elicitators (Radman et al. 2003; Namdeo, 2007). The gaseous phytohormone ethylene has wide ranging effects on plant growth and developmental processes, including senescence abscission, floral transition, fruit ripening, germination and morphogenic response in seedlings (Kieber, 1997). Several line of evidence indicated the stimulating effect of ethylene on diosgenin content and enzymes activities in Trigonella foenum-graecum (Oncina et al. 2002; De and De, 2003; Gómez et al. 2004) and in D. floribunda (De and De, 2005).

Four enzymes, i.e., cycloartenol synthase (CAS), squalene synthase (SQS), farnesyl pyrophosphate synthase (FPPS), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), are well recognized as key enzymes responsible for the biosynthesis of triterpene diosgenin in plants (Figure 1). The synthesis of steroidal sapogenins and alkaloids involves cyclization of squalene 2, 3-epoxide, mediated by cycloartenol synthase, with the formation of tetracyclic, C₃₀-compound- cycloartenol (Brown, 1998). It has been well documented that CAS plays essential roles in the plant cell viability, and in the regulation of triterpenoid biosynthesis. SQS is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene, a key precursor for the sterol and triterpene biosynthesis (Huang et al. 2007; Lee and Poulter, 2008). FPPS is essential for the organ development in plants although it has not previously been identified as a key regulatory enzyme in triterpene biosynthesis (Kim et al. 2010a). The enzyme HMGR catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway (Wang et al. 2007). Accumulating research results suggested that plant HMGRs are encoded by a gene family members and regulated by light, growth regulators, wounding and treatment with pathogen or elicitors (Park et al. 1992; Maldonado-Mendoza et al. 1997).

Several studies have been done to assess the stimulating effects on diosgenin production by ethylene or other elicitors, but the possible genes responsible for the stimulation are largely unknown. The aim of this present work is to understand the molecular mechanism of ethylene induced diosgenin accumulation and the gene expression patterns of CAS, SQS, FPPS, and HMGR in in vitro cultures of D. zingiberensis.

The in vitro cultures were established from Dioscorea zingiberensis C. H. Wright which was originally collected from Wudang Mountain, Hubei Province, China. The cultures were maintained on Murashige and Skoog (1962) salt medium (MS medium) containing 1 mg/L 6-BA, 25 g/L sucrose and 8 g/L agar. The pH of the medium was adjusted to 5.8. The medium was sterilized at 121ºC for 20 min. The tissue cultures were grown and maintained in a culture chamber at 25ºC with a 12 hrs light photoperiod, provided by fluorescent tubes (Philips, 3000Lx); and were subcultured at 4 week intervals.

Diosgenin and BSA were from the Sigma Company. Methanol and ethephon (40% ethylene) were from Fisher Scientific and Sichuan Guo Guang Company, respectively. All other chemicals and reagents used were of the highest purity commercially available. Agilent 1100 workstation was used for HPLC analysis.

After four weeks of subculture, the in vitro cultures were moved into new MS medium which was 6-BA free. Ethephon were diluted 10², 10³, 10⁴, 10⁵ times with sterilized distilled water, and marked as E1 to E4 respectively. The ethephon solution was sterilized by filtration through a sterile 0.22 μm membrane filter. The control samples (CK) were treated with distilled water only. For ethylene addition, 1 cm-wide strips of sterilized filter paper were soaked in elicitor solution and placed on the wall of the culture boxes. Three replicates of 20 boxes (60 plantlets) were done for each treatment. In order to record the initial weight of the materials, the culture bottles were weighed before and after the tissues were moved into the medium. These cultures were incubated under conditions as described above for 40 days before used in further experimental investigations.

The treated and control plantlets were harvested after 40 days culturing, the fresh weight (FW) was determined, and then dried to constant weight at 60ºC for several days and used to measure dry weight (DW). The dried materials were used to assess the diosgenin content.

Preparation of samples and the indirect competitive ELISA assay method was performed as previously described (Li et al. 2010). Diosgenin yields in 10 boxes’ culture were measured using the following formula, Diosgenin yields = diosgenin content (% DW) x DW of 10 boxes culture (mg).

Total soluble proteins were extracted with 100 mM sodium phosphate buffer (pH 7.5) containing 2 mM ascorbate and 0.5 mM ethylene diamine tetraacetic acid (EDTA), and were quantified according to Bradford (1976), using bovine serum albumin as a standard.

The chlorophyll content was measured spectrophotometrically in 80% acetone extracts as described by Arnon (1949). Total chlorophyll (Ct) were calculated using the equation Ct = 1000* A652/34.5.

Cultures treated with ethephon at concentration E3 were used to examine the expression level of genes involved in diosgenin biosynthesis in comparison with the control samples. Total RNA from the treated and control samples, was isolated using TRI plant Total RNA Fast Extraction Kit (Spin-column) (BioTeke Corporation, Beijing) according to manufacturer’s instructions. Fragments containing coding zones of different genes were amplified by RT-PCR. Semi-quantitative PCR reactions were performed using 31 cycles for CAS (GenBank accession no. AM697885), 33 cycles for SQS (GenBank accession no. JN693497), 31 cycles for FPPS (GenBank accession no. JN693498), 28 cycles for HMGR (GenBank accession no. DQ017377) and 27 cycles for actin (GenBank accession no. JN693499), the quantitative primers were designed according to known sequence listed in the Table 1. PCR reactions were carried out in an ABI thermocycler using Taq DNA polymerase (Fermentas) under the following conditions: an initial 5 min denaturing at 94ºC, followed by cycles of 94ºC 20 sec, 58ºC 20 sec, and 72ºC 30 sec. PCR products were sampled at the aforementioned specified cycles and analyzed by 1.5% agarose gel electrophoresis.

Differences between the treatments and the controls of the data were analyzed by an analysis of variance, and the post hoc Duncan’s multiple range test was used when appropriate. Pearson’s correlation test was carried out between diosgenin and other parameters. All statistical analyses were performed using the SPSS package (SPSS Inc., Chicago, IL) and were considered significant at P < 0.05.

Higher concentrations of ethephon (E1, E2, and E3) had inhibitory effect on the growth of cultures (Figure 2), whereas the lower (E4) concentration didn’t induce any evident changes in the treated samples compared with the control samples. The FW decreased in ethephon-treated plants with decrement of 26%, 24%, 14%, and 18% in E1, E2, E3 and E4 compared to CK. Ethephon treatment also significantly lowered dry mass with the highest decrement in E1 (25% compared to CK) and then E3 (23% compared to CK) (Figure 3).

The results (Figure 4a) show that with the decrease of ethephon concentration, the diosgenin level in treated samples temporarily increased and then decreased. The highest level of diosgenin (increase of 149.8% over that of the control samples) was achieved with concentration E3. When expressed with yield, the E3 and E2 showed significant increase (93% and 21% compared to CK) in 10 boxes’ culture plants (Figure 4b).

The accumulation of soluble protein had a similar trend of diosgenin accumulation (Figure 5a). Their levels increased together with diosgenin, which suggests that a determined level of protein accumulation is required for diosgenin biosynthesis in plant tissue. High ethylene concentration inhibited chlorophyll accumulation, whereas plants with E₃ treatment increased chlorophyll content by 33% (Figure 5b).

The results shown in Figure 6 indicated that in comparison with the control samples, the expression of cycloartenol synthase (CAS) gene was up-regulated at all time points, and HMGR gene show significant response to ≥ 8 hrs ethylene treatment. FPPS gene is only slightly up-regulated after 4 hrs elicitor application. However, ethylene treatment didn’t induce any evident changes in the expression of squalene synthase (SQS) gene.

The mechanism of the ethylene effect on the diosgenin synthesis and accumulation is still not clearly understood. This study investigated enhancement effect of ethylene on the accumulation of diosgenin in D. zingiberensis cultures, and the gene expressions of key components involved in diosgenin biosynthesis to delineate the molecular mechanisms governing the process.

The diosgenin biosynthesis and accumulation are very complex processes which are tightly regulated by primary metabolism. Our results showed that the application of certain concentrations of ethylene can improve diosgenin biosynthesis in in vitro cultures of D. zingiberensis (Figure 4) which is consistent with existing findings (De and De, 2003). Recently, Gómez et al. (2004) reported the increase in diosgenin biosynthesis and accumulation when the fenugreek cells suspension was treated with ethephon. The diosgenin content first increased with the decrease of ethephon concentration, and then decreased. The decrease of diosgenin content induced by lower and higher concentration of ethylene may be due to the induction of other metabolic pathways whose activities can inhibit or slow down the diosgenin biosynthesis pathway. In the meantime, a decrease of biomass was observed in ethylene treated cultures (Figure 2, Figure 3), which is in agreement with other reports in Trigonella foenum-graecum (De and De, 2003; Gómez et al. 2004).

The effect of ethylene on diosgenin content is closely related to the soluble protein and chlorophyll content (Figure 5). Diosgenin production is a kind of plant response to stress, in which de novo proteins synthesis is required. Narula et al. (2005) observed the increase of diosgenin and protein content in D. bulbifera callus cultures grown under abiotic stress conditions. Data for ethylene treated and control plants were grouped together to determine the possible correlations between diosgenin and other traits (Table 2). It was showed that diosgenin content was significantly and positively correlated with soluble protein (p < 0.01), as well as chlorophyll (p < 0.01). Ehmke and Eilert (1993) demonstrated a positive correlation between solasodine level and chlorophyll content in suspension cultures of Solanum dulcamara, which is in agreement with our results.

Many studies show that transcriptional modulation of related genes is a common response to pathogens or elicitor signals. Inspections on DNA sequences of elicitors (for example, JA and ethylene) responsive genes have identified several elicitor response elements in these genes which are involved in the biosynthesis of secondary metabolites (Zhao et al. 2005). We provided evidence here that gene expressions of, CAS, HMGR, and FPPS in in vitro cultures of D. zingiberensis were induced by ethylene applications which led to the improvement of diosgenin biosynthesis (Figure 6).

The enzymes CAS are responsible for triterpene biosynthesis catalyzing the formation of cycloartenol which is then converted into cholesterol, the most important precursor of diosgenin (Friedman et al. 1997; Arnqvist et al. 2003). Kim et al. (2011) and Han et al. (2006) reported the elicitor MJ induced up-regulation of CAS in Bupleurum falcatum, and Panax ginseng, respectively. Lee et al. (2004) reported that overexpression of the squalene synthase gene was followed by up-regulation of CAS gene in transgenic P. ginseng. However, Kim et al. (2011) found that methyl jasmonate treatment reduced the expression of CAS gene in the roots of wild Bupleurum falcatum. HMGR, catalyzing the conversion of HMG-CoA to mevalonic acid, is considered as a rate-limiting enzyme in cholesterol synthesis, and it also plays a critical role in controlling isoprenoid derivatives related pathways (Chen et al. 2012). Cools et al. (2011) found that ethylene treatment could increase in the expression of HMGR gene in onion. HMGR gene expression was induced by elicitor in Michelia chapensis (Cao et al. 2011). In our study, the changes in HMGR gene expression seemed to be time-dependent, and the highest level appeared 8 hrs ethylene treatment (Figure 6). FPPS has not been identified as a key regulatory enzyme in triterpene biosynthesis (Kim et al. 2010b). In this work, its expression is time-dependent (Figure 6). Ding et al. (2008) established that FPPS gene possessed various potential regulatory elements associated with physiological and environmental factors. A down-regulation of SQS gene is observed in ethylene treated samples (Figure 6). This finding is similar with what reported by Devarenne et al. (2002), who found that absolute level of the SQS mRNA decreased approximately 5-fold in the elicitor-treated cells, suggesting decreased transcription of the SQS gene.

In conclusion, our results suggest that ethylene treatment enhanced diosgenin accumulation by considerably affecting the expression of CAS and HMGR gene in the present study. Further investigations will be necessary to elucidate the specific role of each gene involved in diosgenin biosynthesis regulation, and to fix the mechanism by which CAS and HMGR can be involved in triterpenes biosynthetic pathway.

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