Accumulation of salicylic acid-elicited alkaloid compounds in in vitro cultured Pinellia ternata microtubers and expression profiling of genes associated with benzoic acid-derived alkaloid biosynthesis
The alkaloid compounds found in Pinellia ternata tubers have major bioactive components, and thus, these plant products are one of the most widely used ingredients in traditional Chinese medicines (TCMs). Under field agricultural growth conditions, however, it usually takes 2 years for tuber formation and growth. In vitro induced microtubers provide an alternative approach for the commercial production of P. ternata tubers for use in the TCM industry. The elicitation effect of supplementation with salicylic acid (SA) on the accumulation of alkaloid compounds in tubers and the related molecular regulation mechanism for biosynthesis are not well understood. In this study, we address this knowledge-gap through the development of an efficient induction system of in vitro cultured microtubers subsequently used to study the mechanism for elicitation of alkaloid compound accumulation by SA. Efficient microtuber induction was achieved by inserting petioles inversely into solid Murashige & Skoog medium (MS) followed by subculturing the morphologically expanded lower portion of the culture petioles in suspension culture, without the additional application of plant growth regulators. The in vitro microtuber induction rate achieved was 100% within 25 days of culture. When treated with 50–150 μM of SA, in vitro cultured microtubers showed higher accumulation of alkaloid compounds over the negative control. The highest accumulation detected showed an increase of 2.5-, 2.1-, 2.8-, and 3.1-fold in the concentration of total alkaloid compounds, guanosine, inosine and ephedrine, respectively, in the presence of 100 μM SA, 15 days after induction. qRT-PCR analysis of candidate genes for key enzymes in alkaloid biosynthesis indicated that CNL, CHY and BALDH are most probably responsible for the accumulation of benzoic acid and other alkaloid derivatives in the in vitro cultured microtubers following SA elicitation. This study developed an efficient in vitro microtuber induction system, and used this to determine that SA-promoted accumulation of alkaloids is associated with genes in the benzoic acid and alkaloid derivative biosynthesis pathway in P. ternata.
Salicylic acid elicitation of the biosynthesis of pharmacologically important alkaloids, such as guanosine, inosine and ephedrine in Pinellia ternata microtubers cultured in vitro without addition of exogenous plant growth regulators. Further expression profiles of biosynthetic pathway genes for intermediate metabolite benzoic acid and alkaloid derivatives suggested possible key synthase genes that are induced in response to SA elicitation.
Alkaloids Elicitation Gene expression profile In vitro microtuber induction
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This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31501368, 81573518, 81803665), the Natural Science Foundation of Anhui Province, China (Grant No. 1608085MC52), the Key Project of Natural Science Research for Universities in Anhui Province, China (Grant Nos. KJ2019A0584, KJ2018A0403); the Innovation Team of Scientific Research Platform of Anhui Province, China (Grant No. KJ2015TD001). The Funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
YD, TX and JX conceived and designed the experiments. TX and JX collected the materials. YD, HZ, XM, MH, ZZ, CH and FZ performed the experiments. YD, TX and JX interpreted the data and wrote the manuscript. All authors reviewed and approved the manuscript.
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