Optimization Of Astragalus Membranaceus Hairy Root Cultures And Elicitation Of Its Main Active Constituent Biosynthesis

Astragalus membranaceus is a promising medicinal plant in the forest of Heilongjiang Province. Usually, A. membranaceus needs to grow 3-5 years before harvesting its roots. Due to the excessive exploition, the wild resources of A. membranaceus have been on the verge of extinction. Although A. membranaceus can be cultivated, the quantities of valuable astragalosides and isoflavonoids are often fluctuating due to unfavorable environmental conditions, which have caused their decline in quality and poor clinical treatment effect. Production of medicinal active components and elucidation of their biosynthesis mechanisms by plant tissue culture technology have become an important research hotspot of plant secondary metabolic engineering and modern forestry biological agent fields. In this work, a rapid and efficient in vitro hairy root induction system of A. membranaceus was successfully established. The high-productive astragaloside (AG) and isoflavonoid (IF) A. membranaceus hairy root lines (AMHRL) were systematically screened and confirmed by PCR amplification. Culture conditions of A. membranaceus hairy roots were systematically optimized by statistical experimental designs and kinetic models, and the main active constituents were quali-quantitatively determined by LC-MS/MS. Subsequently, A. membranaceus hairy root cultures (AMHRCs) were treated by the exogenous physicochemical and biological elicitors. Accumulation patterns of AG and IF before and after elicitor treatments were determined by LC-MS/MS. Expression profiles of enzyme genes involved in AG and IF biosynthetic pathways were monitored by qRT-PCR. Eventually, the key regulated enzyme genes in AG and IF biosynthetic pathways were preliminarily elucidated. The main research contents and results are summarized as follows:1. Optimization of AMHRCs system for the effcient prodcution of AGs and IFs(1) An optimal Agrobacterium rhizogenes LBA 9402-mediated transformation rate (66.84%) was obtained for the induction of A. membranaceus hairy roots when 3 week-old leaf explants were co-cultured for 2 days with the supplementation of acetosyringone (100μM) and then transferred to MS solid medium containing sodium cefotaxim (500 mg/L). Moreover, the duration of hairy root induction was only 13 days under the optimized induction conditions. Based on the biomass production of hairy roots and the accumulation of active ingredients, the high-productive AG root line (AMHRL Ⅵ) and high-productive IF root line (AMHRL Ⅱ) were successfully selected, and confirmed by PCR amplification.(2) Culture parameters of AMHRL VI were systematically optimized by central composite design (CCD) and Slogistic kinetic models. The optimal culture conditions obtained were as follows:culture temperature 27.8℃, inoculum size 1.54%, sucrose concentration 3.24 % and harvest time 36 days. Culture parameters of AMHRCs were systematically optimized by Box-Behnken design (BBD). The optimal operational conditions were obtained as follows: culture temperature 27.8℃, inoculum size 1.44%, sucrose concentration 3.39% and harvest time 34 days. Six AG derivatives and five IF constituents were quali-quantitatively determined by LC-MS/MS. Under the optimal conditions, AMHRCs pproduced total AG (2.657±0.088 mg/g DW) and total IF (234.77±4.27μg/g DW), which were significantly superior to those of 3 year-old field grown A. membranaceus roots (2.435±0.093 mg/g DW and 187.38μg/g DW).2. Regulation of AG and IF biosynthesis in AMHRCs by the elicitation of plant signal moleculars(1) In this work, the elicitation of AMHRCs with methyl jasmonate (MJ), salicylic acid (SA) and acetylsalicylic acid (ASA) was conducted to boost AG and IF biosynthesis. MJ was found to be the most effective inducer for AG and IF production among all elicitor treatments. MJ elicitation conditions were systematically optimized by CCD. The highest enhancement of AG accumulation was achieved in 34 day-old AMHRCs VI elicited by 157.4μM MJ for 18.4 h. The resulting AG yield was 5.547±0.133 mg/g dry weight (DW), which was 2.07-fold higher than that of non-treated control (2.685±0.051 mg/g DW). The optimal enhancement of IF production was obtained in 34 day-old AMHRC II elicited by MJ under the concentration of 283μM and the exposure time of 33.75 h. The resulting IF yield was 2250.10±71.88μg/g dry weight (DW), which tremendously increased 9.71-fold relative to that of non-treated control (231.64±6.51μg/g DW).(2) Transcriptional levels of nineteen genes involved in AG and IF biosynthetic pathways were assayed by qRT-PCR. Accumulation patterns of AG and IF were detected by LC-MS/MS. A relationship between gene transcription and active ingredient content was established for preliminarily clarifying the regulation governing points in AG and IF biosynthetic pathways. Results showed that IDI, MVD, FPS and SS were the potential key enzyme genes controlling AG biosynthesis in response to MJ elicitation. Also, CHI and IFS were found to be crucial regulatory enzyme genes in IF biosynthetic pathway following MJ elicitation. Moreover, activities of antioxidant enzymes in AMHRCs as well as antioxidant effects of extracts from AMHRCs were siginificantly enhanced under MJ elicitation, which demonstrated that AMHRCs showed a significant oxidative stress defense in response to MJ elicitation.3. Regulation of AG and IF biosynthesis in AMHRCs by the elicitation of UV radiation(1) In this work, AMHRCs were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting AG and IF biosynthesis. UV-B was found to be the most effective inducer for AG and IF production among all elicitor treatments. The treatment of 32 day-old AMHRCs VI with 54 kJ/m2 dose of UV-B radiation was capable of achieving the maximum production of AGs, i.e.3.431±0.092 mg/g DW, which was 1.30-fold higher as compared to the level in non- treated control (2.645 ± 0.073 mg/g DW). The optimum enhancement for isoflavonoid production was achieved in 34 day-old AMHRCs elicited by 86.4 kJ/m2 of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g DW, which was 2.29-fold higher relative to control (232.93 ± 3.08μg/g DW).(2) Transcriptional levels of nineteen genes involved in AG and IF biosynthetic pathways were assayed by qRT-PCR. Accumulation patterns of AG and IF were detected by LC-MS/MS. A relationship between gene transcription and active ingredient content was established for preliminarily clarifying the regulation governing points in AG and IF biosynthetic pathways. Results showed that HMGR was the only potential key enzyme gene controlling AG biosynthesis in response to UV-B elicitation. Also, PAL and C4H were found to be two crucial regulatory enzyme genes in IF biosynthetic pathway following UV-B elicitation. Moreover, activities of antioxidant enzymes in AMHRCs as well as antioxidant effects of extracts from AMHRCs were siginificantly enhanced under UV-B elicitation, which demonstrated that AMHRCs showed a significant oxidative stress defense in response to UV-B elicitation.4. Deacetylation biocatalysis and biotic elicitation of immobilized Penicillium canescens (IPC) in AMHRCs for the enhanced production of astragaloside Ⅳ (AG Ⅳ)(1) In this work, a special fungus P. canescens was found to possess strong deacetylation ability that can effectively transform other AG derivatives to AG Ⅳ. The immobilization of P. canescens spores in Ca-alginate gel beads was sucessfully prepared. A co-cultivation system of AMHRCs and IPC was established for the enhanced production of AG Ⅳ. On one hand, the deacetylation biocatalysis of IPC could achieve the biotransformation of some AG Ⅳ precursors (astragaloside Ⅰ, astragaloside Ⅱ, isoastragaloside Ⅱ) to AG IV with high-efficiency. On the other hand, the elicitation of AMHRCs by IPC can also trigger plant defense metabolisms for further promoting AG IV production.(2) The optimal deacetylation conditions of IPC in AMHRCs were obtained as follows: 105 spores per flask, co-cultivation temperature 30℃, pH 7.0 and co-cultivation time 60 h. Under the optimal conditions, astragaloside Ⅰ, astragaloside Ⅱ and isoastragaloside Ⅱ were almost completely transformed to AG Ⅳ by IPC deacetylation. Moreover, transcriptional analyses revealed that expression levels of eight genes involved in AG Ⅳ biosynthetic pathway (AACT, HMGS, HMGR, MK, FPS, SS, SE and CAS) were significantly up-regulated in response to IPC elicitation. By the aid of deacetylation and elicitation of IPC, the resulting AG IV yield was 2.816±0.052 mg/g DW, which tremendously increased 14.59-fold higher than non-treated control (0.193 ± 0.007 mg/g DW). Additionally, the total IF yield in IPC-treated AMHRCs was 673.21 ± 10.75μg/g DW, which was 2.83-fold higher as against control (236.97 ±3.50 μg/g DW). Meanwhile, all IF biosynthetic genes (PAL, C4H,4CL, CHS, CHR, CHI, IFS and 13’H) were considerably induced by IPC elicitation. Furthermore, IPC possesses excellent stability, which could keep its deacetylation activity and elicitation effect stable during the storage period of 60 days. After six recycles, IPC could still maintain its good deacetylation activity and elicitation effect with the integrity of sphere shape. This study provided valuable data for the further industrial production of AG Ⅳ using this technique.Astragalosides and isoflavonoids are not only plant active secondary metabolites, can also act as as phytoalexins/phytoanticipins that can protect plant from environmental stresses. Elucidation of key enzyme genes in their biosynthestic pathways is benifical for cultivating new medicinal plant variety with stress resistance, and also pave the way for astragaloside and isoflavonoid biosynthesis via plant metabolic engineering strategies in the future.