Identification of acyl transferases responsible for the diverse hydroxycinnamoyl chemical space in bidens pilosa

Abstract

For decades, plants have been the backbone of complicated traditional herbal medicine system. Plants have been used by people and animals as a source of nutrients as well as medicine. Production of secondary metabolites by these plants is a characteristic that makes them attractive to both animals and humans. In plants, secondary metabolites play a role in defence mechanism and assist the plant to adapt to their immediate environment. Secondary metabolites are known to possess anti-diabetic, anti-malaria, anti-inflammatory and alleviate complications associated with obesity and cardiovascular diseases. Plants from the Asteraceae family are known to contain metabolites with nutraceutical properties. Plants such as Helianthus annuus, Lactuca sativa, Chicorium intybus and Bidens pilosa are some of the edible examples within the Asteraceae family known to exhibit interesting nutraceutical properties. B. pilosa adapt to almost every environmental condition, which makes it to be found in all parts of the world. As such, this plant has been used to manage and treat illnesses affecting humankind. Unique to this plant is the existence of large contingency of structurally diverse chlorogenic acids. B. pilosa is known to produce different structural hierarchies of chlorogenic acids (i.e., mono-, di-, tri- acyls). The biosynthetic pathway to produce the diverse array of chlorogenic acids in B. pilosa is not yet elucidated. It is known from other plants like Helianthus annuus that the production of chlorogenic acids is coded by hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase gene (HQT) and hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT). Apart from the chlorogenic acids (quinic acid acyls), B. pilosa is known to also produce acyls of tartaric acid, also characterised by existence of structurally diverse isomers thereof. From other plants, the tartaric acid esters are coded by the hydroxycinnamoyl-CoA: tartaric hydroxycinnamoyl transferase (HTT) gene and, surprisingly, the gene encoding the tartaric acid esters/acyls from B. pilosa is also not known. It is therefore imperative that a study aimed at establishing/identification of the gene elements which are responsible for the diversification of chlorogenic acids and related compounds (such as tartaric acid acyls) in B. pilosa is conducted. To achieve this, a Single Molecule Real Time (SMRT) sequencing approach was used to establish the full-length gene transcripts, in attempt to identify the acyl transferase Executive summary xi responsible for chlorogenic acids production in B. pilosa. The SMRT sequencing technique has brought a lot of improvement from the Sanger and Next generation sequencing such as generation of long reads which overcomes the challenges of sequence assembly synonymous with the short reads achieved by the former two sequencing approaches. Moreover, this technique allows detection of isoforms of a specific gene, caused by either inherited genetic code or alternative splicing events. From the SMRT sequencing results, three HQT genes and one HCT gene responsible for production of wide array of chlorogenic acids and two HTT genes responsible for production of tartaric acid esters were identified through series of bioinformatics analyses of the sequences obtained through SMRT sequencing. All the identified genes contained the conserved regions that are found in already published acyltransferases, with the highly conserved DFGWG motif present in all transcripts identified herein. The second motif, the HXXXD motif showed a single amino acid variation from gene to gene, with HQT1 and HQT2 showing HTLSD motif and HQT3 having HTLAD motif, all of which are synonymous with the plants from the Asteraceae family. In HTT genes, the second motif identified in B. pilosa has never been recorded in literature. HTT1 and HTT2 showed to have HRVLD and HRVAD motif respectively. From these sequences, the open reading frames (ORFs) were computed, and these sequences can be used to design primers that can be used to amplify these genes in the future. Through multi sequence alignment and phylogenetic trees, the identified genes were also found to have similarities with the genes of other plants from the Asteraceae family. In conclusion, the SMRT sequencing approach enabled identification of acyltransferases genes that plays a role in the biosynthesis of CGAs in B. pilosa. Bioinformatics tools were shown to be sufficient to annotate and characterise these genes. Through LC-MS analyses of randomly collected B. pilosa plants, the CGAs content of this plant were revisited, and these plants were found to produce structurally diverse CGAs compounds, suggesting that the identified genes are functional. Future studies should aim to clone these transcripts in plant systems that do not produce CGAs in attempt to enhance their nutraceutical attributes.