SMRT sequencing and characterization of putative hydroxycinnamoyl-CoA: Quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes responsible for biosynthesis of structurally diverse chlorogenic acids in Bidens pilosa

Abstract

Bidens pilosa L. is an underutilised plant that serves as an ingredient for traditional herbal medicine and as food. This plant has received considerable attention because of its chemistry, which comprises of chemicals from various classes, with those from the phenylpropanoid pathway (e.g., chlorogenic acids) existing in abundance. B. pilosa L. produces chemically diverse chlorogenic acids (CGAs), characterised mainly by isomers thereof. The enzymes involved in the biosynthesis of chlorogenic acids have been decoded and characterised in many plants. However, genes that play a role in the biosynthesis of chlorogenic acids in B. pilosa L. are not yet described and, as such, the aim of the current study was to identify different isoforms of the Hydroxycinnamoyl-CoA: quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes that play a role in the diversification of chlorogenic acids in Bidens pilosa L. Herein, a robust gene sequencing technology through Single Molecule Real Time (SMRT) approach was applied to establish the gene sequences encoding transferases responsible for CGA production in B. pilosa L. Sequence homology of the established genes was evaluated by means of multiple sequence alignment against already published orthologous genes from closely related plants. Thereafter, phylogeny trees were constructed to graphically display the taxonomical relationship with other chlorogenic acid producing plants. To further demonstrate the functionality of these genes in plants, chlorogenic acid profiling was carried out using Liquid chromatography quadrupole time of flight mass spectrometer (LC-QTOF-MS). B. pilosa L. plants produced isomeric forms of mono, di and tri-acylated CGAs. A total of three HQT genes and one HCT gene have been identified in this study and have been shown through homology to be related with other related genes from other CGAs producing plants. Future work should involve expression of the identified genes in other non-chlorogenic acid producing plants to enhance their nutraceutical value.