Elicitation of Phenyl Propanoid production and expression profilint of Acteoside Biosynthetic Genes in Artanema sesamoides Benth (Vathomv Arettii)

Abstract

The study entitled “Elicitation of phenyl propanoid production and expression profiling of acteoside biosynthetic genes in Artanema sesamoides Benth (vathomvaretti)” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objectives were to study the effect of elicitors like salicylic acid, methyl jasmonate, abscisic acid and yeast extract on phenyl propanoid glycosides production in A. sesamoides and to analyse the expression profile of key genes of acteoside biosynthesis pathway, such as PAL (phenylalanine ammonia-lyase), HCT (Shikimate O hydroxycinnamoyl transferase) and UGT (UDP glucose glucosyl transferase). Callus culture was established in MS medium supplemented with 0.5 mgL1 BA and 0.5 mgL-1 NAA using in vitro plants of A. sesamoides. Two week old callus cultures were transferred to liquid MS medium with 0.5 mgL-1 NAA and 0.5 mgL-1 BA and stabilized for 10 days. Elicitors viz., salicylic acid (SA; 40 and 100µM), methyl jasmonate (MJ; 15 and 25µM), abscisic acid (ABA; 20 and 50µM) and yeast extract (1 and 1.5 gL-1) were added to the cultures and incubated in orbital shaker (120 rpm) at 24 °C under dark condition for 48h. After elicitation, callus was harvested, dried and finely powdered. Phenyl propanoid glycosides were extracted by sequential extraction using solvents like hexane, chloroform and methanol. Methanol extracts were collected, purified and evaporated to get the residues. The total content of phenyl propanoid glycosides was maximum (4.160 mg/g dry weight) in callus treated with yeast extract (1.5 gL-1). The residues re-dissolved in HPLC grade methanol were analysed by HPLC using PDA detector at different wavelengths (197, 218, 254 & 330 nm). Six important PPGs viz., acteoside, artanemoside, isoacteoside, leucosceptoside, martynoside and plantainoside were identified from the methanol extract. The highest peak of HPLC chromatogram representing the most prominent antioxidant 87 phenyl propanoid glycoside (acteoside) in the extract appeared within the retention time of 59 min. YE (1gL-1) has significantly increased the content of all the major PPGs. The maximum content of acteoside (36.951%) and isoacteoside (22.220%) was obtained in calli treated with yeast extract (1.5 gL-1). The effect of elicitors on expression profile of acteoside biosynthesis genes such as PAL, HCT and UGT was analysed 24h and 48h after elicitation by real time PCR using SYBR® Green dye. Primers were designed using “Primer Express” software. RNA isolated from the callus after 24 and 48h of elicitation was converted to cDNA and the quality was confirmed by PCR using ACTIN gene specific primers. Cq values obtained in RT-qPCR for each gene was analysed using “qbase plus” software with ACTIN as the reference gene. Expression of PAL gene which acts in the upstream of acteoside biosynthetic pathway was found upregulated by all the elicitors. The highest elicitation (26 fold) was shown by SA (40μM), followed by yeast extract (18 fold with 1.5 gL-1), MJ (16 fold with 25μM) and ABA (11 fold with 50μM). Treatment with SA produced upregulation of PAL at 24h, while all other elicitors enhanced its expression at 48h. All the elicitors enhanced the expression of UGT at 48h of elicitation. The highest expression (42 fold) was with SA (100μM), followed by yeast extract (20 fold with 1 gL-1) and MJ (10 fold with 25μM). In this study, the two key genes, PAL and UGT, involved in the acteoside biosynthetic pathway were upregulated by all the elicitors. But only yeast extract significantly enhanced the in vitro production of six major phenyl propanoid glycosides (artanemoside, isoacteoside, leucosceptoside, martynoside and plantainoside) in A. sesamoides. The study shows a possible use of yeast extract in the in vitro production of phenyl propanoid glycosides.