Abstract
The plant Ophiorrhiza pumila produces camptothecin (CPT), a kind of terpene indole alkaloid (TIAs) that has been widely used in treatment of cancer. Tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been reported to play important roles in plant metabolism and development. In this study, a novel WRKY transcription factor named OpWRKY3 was isolated from O. pumila, with full-length open reading frame (ORF) of 1128 bp, encoding 375 amino acids. Phylogenetic tree analysis revealed that OpWRKY3 shared the highest homology with VvWRKY30, and it is a significant feature belonging to group III. OpWRKY3 was responsive to various treatments, including gibberellin (GA3), methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA). Besides, OpWRKY3 is expressed predominantly in stems. Subcellular localization analysis showed that OpWRKY3 localized in the nucleus. The biomass of OpWRKY3-SRDX transgenic hairy roots (S line) was visibly suppressed, while there were slight changes between overexpression of the OpWRKY3 line (OE line) and the control. In addition, the concentration and total production of camptothecin precursors including loganin and secologanin were significantly changed in both OE and S lines while total production of CPT was significantly changed in most transgenic lines. Thus, the present work revealed that OpWRKY3 may act as a regulator in the growth and development of O. pumila, and in production of camptothecin and its precursors.
Highlights
Camptothecin (CPT) is a well-known terpene indole alkaloid (TIA), which was isolated for the first time by M
To investigate whether OpWRKY3 responded to exogenous elicitor treatment, 30-day old hairy roots were treated with different inducers, including GA3, methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA)
The results showed that the exogenous GA3, MJ, or ASA significantly induced OpWRKY3 expression, reaching a peak at 1 h (Figure 2B–D), and thereafter declined rapidly
Summary
Camptothecin (CPT) is a well-known terpene indole alkaloid (TIA), which was isolated for the first time by M. Geranyl pyrophosphate (GPP) was catalyzed by a series of enzymes, including geraniol synthase (GES), geraniol-8-oxidase (G8O), 8-hydroxy-geraniol oxidoreductase (8-HGO), iridoid synthase (IS), iridoid oxidase (IO), 7-deoxyloganetic acid UDP-glucosyltransferase (7-DLGT), 7-deoxyloganic acid hydroxylase (7-DLH), loganic acid O-methyltransferase (LAMT), geraniol-10-hydroxylase (G10H), 10-hydroxy-geraniol oxidoreductase (10-HGO), and secologanin synthase (SLS), forming secologanin [5,11] In this pathway, the enzyme nicotinamide adenine dinucleotide phosphate (NADPH): cytochrome P450 reductase (CPR) is essential for the activity of G10H and SLS, which plays a vital role in electron transfer from NADPH to cytochrome P450 [12]. Previous studies demonstrated that WRKY transcription factors (TFs) play an important role in multiple physiological activities, including plant growth and development, senescence, biotic and abiotic stress responses, secondary metabolism, and phytohormone signaling [26,27,28,29]. The findings of this study will serve as an accentuating reference for future research on the regulators of alkaloid production in various medicinal plants
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