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. 2024 Feb 13;22(1):100344. doi: 10.1016/j.jgeb.2023.100344

Characterization of phenylalanine ammonia lyase and revealing flavonoid biosynthesis in Gymnema sylvestre R. Br through transcriptomic approach

Kuldeepsingh A Kalariya 1,⁎, Ravina R Mevada 1, Manish Das 1
PMCID: PMC10903758  PMID: 38494263

Abstract

Background

Gymnema sylvestre R.Br. is famous medicinal plant among diabetics for its gymnemic acid content. It also contains flavonoids, which are an essential component in various other products. Though some molecular information on the biosynthesis of gymnemic acid, polyoxypregnane, micro RNAs and photosynthetic efficiency is available, there is no gene level information available on the biosynthesis of flavonoids in this plant. RNA was extracted from winter-collected Gymnema sylvestre leaves and cDNA libraries were prepared and used for next generation sequencing. De novo transcriptome assembly were prepared and Coding DNA Sequences (CDS) of 13 major genes involved in flavonoids biosynthesis were identified from transcriptome data. Phenylalanine ammonia lyase gene containing full-length CDS was employed for in silico protein modelling and subsequent quality assessment. These models were then compared against publicly available databases. To confirm the identification of these genes, a similarity search was conducted using the NCBI BLAST tool.

Results

Therefore, in the present study, an effort has been made to provide molecular insights into flavonoid biosynthesis pathway by examining the expressed transcripts in G.sylvestre. Gene sequences of total thirteen major genes viz., phenylalanine ammonia lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, shikimate O-hydroxycinnamoyl transferase, coumaroyl quinate (coumaroyl shikimate) 3′-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, naringenin 3-dioxygenase, flavanol synthase, flavonoid 3′-monooxygenase, Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase and leucoanthocyanidin dioxygenase were identified and a putative pathway of flavonoids biosynthesis has been illustrated based on transcriptome data.

Conclusions

This transcriptome study has contributed gene-level insights into the biosynthesis of flavonoids in plants as a whole and represents the first report within a non-model plant, Gymnema sylvestre perticullarly.

Keywords: Flavonoid biosynthesis, Gymnema sylvestre, Phenylalanine Ammonia Lyase, RNA sequencing, Transcriptome analysis

1. Background

Gymnema sylvestreR.Br. is renowned for its effectiveness in managing diabetes. This fame is primarily attributed to the medicinal properties of gymnemic acids found in its leaves. In addition to gymnemic acids, this plant also contains flavonoids.1 The flavonoids are famous for their beneficial effects on human health. They have now become essential components in various nutraceutical, pharmaceutical, medicinal and cosmetic products.2 These flavonoids comprise a group of water-soluble phenylpropanoids stored within the vacuoles of plant cells.3 All classes of flavonoids share a common basic carbon skeleton structure, known as C6-C3-C6, with the exception of stilbenes, which have a C6-C2-C6 structure. This fundamental structure consists of two 6-carbon benzene rings (referred to as rings A and B) connected by a 3-carbon heterocyclic ring (referred to as ring C).4 The attachment of B ring on the carbon of the C ring and the oxidation and unsaturation of the C ring classify the flavonoids into subgroups. Linkage of B ring in position 3 and 4 of the C ring makes isoflavones and neoflavanoids, respectively. Several subgroups namely anthocyanins, catechins, chalcones, flavanols, flavanones, flavanonols, flavones and flavonols etc., are formed based on structural features of the C ring and when the B ring is linked in position 2.5 Glycosylation, acylation and various types of other modifications creates a large number of flavonoid compounds.6, 7

Survey of literature indicates that the number of plant flavonoids isolated and identified are approaching the five-digit mark. Recent publications have provided comprehensive descriptions of flavonoid biosynthesis in plants.8 Quercetin and kaempferol are flavonoids that have been identified in G. sylvestre through chromatographic and spectral studies.9 Myricetin and furocoumarins, also reported in this plant contributing to its pharmacological benefits, highlighting the potential role of flavonoids as antioxidants. Flavonoids in plants are produced through the shikimate pathway, starting from phenylalanine as a precursor.10 Key enzymes such as phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL) have a significant role in the initiation of the production of p-coumaroyl-CoA. Subsequent steps in the biosynthesis of flavonoids involve the additional enzymes, including chalcone synthase (CHS),11 Chalcone reductase (CHR), chalcone 2′-glucosyltransferase (CH2′GT), chalcone isomerase (CHI), flavonol synthase (FLS), 3β-hydroxylase (F3H) and flavone synthase I (FNS I).12

NCBI SRA data repository search revealed that nearly 80bB raw data of transcriptome in G. sylvestre is published. Based on refined and processed data from this raw data, micro RNAs are identified13 and SQS gene is characterized.14 The transcriptome-level variation in photosynthetic efficiency among different genotypes of Gymnema sylvestre has been discussed. The putative pathway of biosynthesis of gymnemic acid15 and polyoxypregnane16 are also deciphered based on transcriptome data in G. sylvestre. However, there is a lack of available information regarding the biosynthesis of flavonoids in this plant. Therefore, the current study was undertaken to discover the pathway for flavonoid biosynthesis through de novo transcriptome analysis using the Illumina next-generation sequencing platform. The present study denotes the first effort to explore molecular insights into the biosynthesis of flavonoids by examining the expressed transcripts in Gsylvestre.

2. Methods

The leaves of G. sylvestre collected during winter season and the RNA was used for cDNA synthesis. The entire process of transcriptome analysis followed the methodology outlined in a recent transcriptome study conducted on Gymnema sylvestre.17 The CDS representing component genes of flavonoid biosynthesis were identified from this transcriptome analysis and gene with full-length CDS were used for Insilco protein modelling, quality analysis of modelled protein and similarity of the proteins with available published data bases. The phenylalanine ammonia lyase (PAL) protein model was generated through the SWISSMODEL online module using Expasy web server18 and various quality indices were recorded in line of recent transcriptome study in G. sylvestre.14 The CDS sequences encoding PAL in G. sylvestre were blasted for similarity search using the NCBI searched module and the ten most similar sequences were downloaded and used to study the similarity at nucleotide sequences level. Using the Mega 11.0 module, the phylogeny tree was prepared keeping test of phylogneny as bootstrap and a number of bootstraps as 1000. The chosen model was the Tamura-Nei model, which incorporated all sites for gaps/missing data treatment and the analysis was performed with three threads. Correspondingly, the similarity search was carried out for scaffolds representing these 13 identified flavonoid biosynthesis pathway genes against NCBI’s non-redundant (NR) protein database using the blastX algorithm and sequence similarity was explained.

3. Results

The comprehensive information about the transcriptome size, its quality, numbers of transcripts, CDSs and their quality are available in the published article.15

3.1. Characterization of phenylalanine ammonia lyase (PAL) gene in G. sylvestre

Transcriptome analysis revealed that the phenylalanine ammonia lyase (PAL, EC:4.3.1.24) in G. sylvestre is comprised of 2157 bases. The CDS encoding PAL gene is available at NCBI Nucleotide repository with GenBank accession no. OQ190562. These sequences were used to search for a conserved domain of the gene using NCBI’s conserved domain search against the NCBI curated database. The value threshold was kept 10−2 and a maximum number of hits to be 500 allowing composition-based statistics adjustment. The search revealed the CDS sequences from G. sylvestre gave specific hits with the domain PLN02457 with an interval of 19—2154 and E-value of 0e+00. The consensus CDS encoding protein sequence of the GS-PAL was used to predict the protein model through SWISSMODEL online window (Fig. 1). The model generated was homo-tetramer with MolProbity Score and Clash Score of 1.15 and 1.80, respectively. The QMEAN and Cβ value of the protein were −0.86 and 0.46, respectively. Ramachandran Z-score of the protein was −1.877 describing how well each residue fitted into the allowed areas of the Ramachandran plot (Fig. 2). The phylogeny tree revealed that GS-PAL shows maximum similarity with PAL of Morinda critifolia (Fig. 3).

Fig. 1.

Fig. 1

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Protein model of GS-PAL generated through SWISSMODEL online module. GS-PAL: Gymnema sylvestre- phenylalanine ammonia-lyase.

Fig. 2.

Fig. 2

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Ramchandran Plot analysis of modelled protein.

Fig. 3.

Fig. 3

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Phylogenetic analysis GS-PAL gene based on Maximum Likelihood (ML) method.

3.2. Genes involved in flavonoid biosynthesis in Gymnema sylvestre R. Br

A putative pathway of flavonoid biosynthesis in G. sylvestre illustrated based on genes identified from the transcriptome data is presented in Fig. 4.

Fig. 4.

Fig. 4

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Putative pathway of flavonoid biosynthetic in G. sylvestre. i. C4H: Trans-Cinnamate 4-monooxygenase [EC:1.14.13.11] (MT900543). ii. HST: Shikimate O-hydroxycinnamoyl transferase [EC:2.3.1.133] (MT900547). iii. coumaroyl quinate (coumaroyl shikimate) 3′-monooxygenase [EC:1.14.13.36] (MT889455). iv. CCOAOMT: Caffeoyl-CoA O-methyltransferase [EC:2.1.1.104] (MT900548). v.CHS: Chalcone synthase [EC:2.3.1.74] (MT900539). vi. CHI: Chalcone isomerase [EC:5.5.1.6] (MT900540). vii. F3H: Naringenin 3-dioxygenase [EC:1.14.11.9] (MT900541). viii. FLS: Flavanol synthase [EC:1.14.11.23] (MT900542). ix. F3′H: Flavonoid 3′-monooxygenase [EC:1.14.13.21] (MT900544). x. C12RT1: Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase [EC:2.4.1.236] (MT900546). xi. LDOX: Leucoanthocyanidin dioxygenase [EC:1.14.11.19] (MT900545).

3.3. 4-coumarate: CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (C4H) genes in G. sylvestre

The genetic identification of PAL, C4H and 4CL, the crucial enzymes in the biosynthesis of phenylpropanoids has been accomplished. The enzyme catalysing the formation of p-coumaroyl-CoA and the CDS encoding 4-coumarate: CoA ligase (4CL, EC:6.2.1.12) in G. sylvestre consists of 1650 bases and is available in the NCBI Nucleotide repository with GenBank accession no. OQ190563. The CDS encoding trans-cinnamate 4-monooxygenase (C4H) in G. sylvestre is published with GenBank accession no. MT900543 at the NCBI Nucleotide repository (Fig. 4).

3.4. Shikimate O-hydroxycinnamoyl transferase (HST), caffeoyl-CoA O-methyltransferase (CCOAOMT), chalcone synthase (CHS) genes in G. sylvestre

The CDS encoding the enzyme shikimate O-hydroxycinnamoyl transferase (HST, EC:23.1.133) involved in lignin biosynthesis within the flavonoid biosynthetic pathway in G. sylvestre is available in the NCBI nucleotide with the GenBank accession no. MT900547. This conversion is catalyzed by the enzyme coumaroyl quinate (coumaroyl shikimate) 3′-monooxygenase (EC:21.1.104), which is encoded by the CDS available in the NCBI nucleotide database with the GenBank accession no. MT889455 (Fig. 4). The CDS encoding for caffeoyl-CoA O-methyltransferase (CCOAOMT, EC:21.1.104) is involved in catalysing the feruloyl-CoA synthesis from caffeoyl-CoA further. The enzyme CCOAOMT is available in NCBI nucleotide repository with GenBank accession no. MT900548. These CoAs, the precursor of flavonoid biosynthesis pathway was involved in chalcone catalyses to form the first intermediate metabolite of flavonoid biosynthesis, chalcone through the chalcone synthase (CHS, EC:2.3.1.74), which is encoded by the CDS available in the NCBI’s nucleotide sequences data base with GenBank accession no. MT900539. The CHS in G. sylvestre is made up of 1176 bases. The sequences when blasted showed specific heat (e value = 1.20e-161) with chalcone and stilbene synthases; plant-specific polyketide synthases (PKS) and related enzymes, also called type III PKSs.

3.5. Chalcone isomerase (CHI), naringenin 3-dioxygenase (F3S), flavanol synthase (FLS), flavonoid 3′-monooxygenase (F3′H), flavanone 4-reductase (FNR) and leucoanthocyanidin dioxygenase (LDOX) in G. sylvestre

The enzyme creating heterocyclic ring C in the flavonoid pathway, chalcone isomerase (CHI, EC: 5.5.1.6) in G. sylvestre is consisting of 696 nucleotide bases and it is available at NCBI GenBank with accession no. MT900540. The CDS encoding naringenin 3-dioxygenase (F3S, EC:1.14.11.9) and CDS encoding flavanol synthase (FLS, EC:1.14.11.23) with GenBank accession no. MT900541 and MT900542, respectively are also members of the flavonoid biosynthesis pathway in G. sylvestre through catalysing the oxidation of dihydroflavonols to flavonols making a double bond between C-2 and C-3 of the C-ring (Fig. 4). The CDS encoding flavonoid 3′-monooxygenase (F3′H, EC:1.14.13.21) in G. sylvestre is published with GenBank accession no. MT900544 has been involved in the production of myricetin from either kaempferol or quercetin. Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase (C12RT1, EC:2.4.1.236) is a similar of flavanone 4-reductase (FNR) identified in G. sylvestre. The CDS encoding C12RT1 is available at NCBI GenBank with accession no. MT900546. The CDS sequence of leucoanthocyanidin dioxygenase (LDOX, EC:1.14.11.19) in G. sylvestre is published with GenBank accession no. MT900545. This LDOX has been responsible for production of anthocyanidins.

4. Discussion

4.1. Phenylalanine ammonia lyase (PAL) gene in G. sylvestre

In plants, flavonoids are produced from phenylalanine, which is synthesized via the shikimate pathway, using the phenylpropanoid pathway.10 The search at the Conserved Protein Domain tool indicated that the Family PLN02457 belongs to the superfamily of Conserved Protein Domain Family Lyase I-like. The enzymes, phenylalanine ammonia-lyase and histidine ammonia-lyase fall under the Lyase class I-like superfamily and are functional in catalyzing beta-elimination reactions in plants as active homo-tetramers. Each of the four active sites in the homo-tetrameric enzyme is constituted by residues originating from three distinct subunits. MolProbity, a web-based structure-validation service, offers a comprehensive and well-founded assessment of model quality, encompassing both global and local aspects for both proteins and nucleic acids. The score expressing how well the backbone conformations of all residues correspond to the known allowed areas in the Ramachandran plot carried out for the identified CDS (OQ190562) was within the expected ranges for well-refined structures.

Previously, the association between the concentrations of anthocyanins and other phenolic compounds and the activity of PAL in strawberry fruit has been observed.19 The general phenylpropanoid pathway has three initial steps.3 In this pathway, an aromatic amino acid phenylalanine is converted to p-coumaroyl-CoA. This transformation is facilitated by the activity of PAL, C4H and 4CL. In the first devoted step in this process, Phenylalanine is deaminated by PAL and forms trans-cinnamic acid20 and furthermore, it plays a role in redirecting carbon flow from primary to secondary metabolism in plants.21 In the second step, hydroxylation of trans-cinnamic acid to generate p-coumaric acid which is also the first oxidation reaction in the flavonoid synthesis pathway22 is mediated via the activity of C4H, a cytochrome P450 monooxygenase in plants. PAL-encoding gene; StlA has been involved in the production of the stilbene antibiotic.20

4.2. 4-coumarate: CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (C4H) genes in G. sylvestre

During the third step, the enzyme 4CL catalyses the addition of a co-enzyme A (CoA) unit to the p-coumaric acid resulting in formation of p-coumaroyl-CoA. Notably, the 4CL gene exhibits a distinct substrate-specific action. In Arabidopsis thaliana, At4CL1, At4CL2 and At4CL4 are implicated in lignin biosynthesis whereas At4CL3 is associated in flavonoid metabolism.23 Expression of PAL, C4H and 4CL is frequently observed to be in coordination with each other24 and in plants, the activity of 4CL is positively associated with the anthocyanin and flavanol content in response to stress.25 The p-coumaroyl-CoA gets synthesised from cinnamoyl-CoA with the help of C4H enzyme. This enzyme plays a role in phenylalanine metabolism and phenylpropanoid biosynthesis by converting trans-cinnamate into p-coumarate. Flavonoid biosynthesis leads to the production of particular flavonoids, using the end product of the phenylpropanoid pathway, p-coumaroyl-CoA. The lignin content in A. thaliana was found to correlate with the expression level of the C4H gene.3

4.3. Shikimate O-hydroxycinnamoyl transferase (HST), caffeoyl-CoA O-methyltransferase (CCOAOMT), chalcone synthase (CHS) genes in G. Sylvestre

The HST plays a role in the conversion of p-coumaroyl-CoA to caffeoyl-CoA by converting p-coumaroyl shikimic acid to caffeoyl shikimic acid and p-coumaroyl-quinic acid to caffeoyl quinic acid. These CoAs serve as precursors in the pathways of flavonoid biosynthesis and play a role in chalcone catalysis to produce a variety of flavonoids. The first Intermediate metabolite in flavonoid biosynthesis is the chalcone.4 The acetyl-CoA carboxylase (ACCase) produces malonyl-CoA. Naringenin chalcone is produced from one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA through the action of CHS.11 Depending on the nature of the starter molecule, the Plant-specific Polyketide Synthase (PKS) generate an array of different products. These PKSs, categorized within the superfamily PLN03173 are dimeric iterative PKSs that utilize CoA esters to transport substrates to the active site. However, they vary in their in the choice of starter molecule and the number of condensation reactions. A SWISSMODEL model prediction of the protein sequences revealed it to be a homo-dimer with 78.96 % sequence identity with Chalcone synthase 1, Crystal structure of Type III polyketide synthase from Oryza sativa template ID 4yjy.1. The model generated was homo-dimer with MolProbity Score and Clash Score of 1.02 and 1.09, respectively. Ramachandran plot analysis indicated Ramachandran Favoured score of 96.74 % and the outliers on chain A included proline (A306), serine (A91), valine (A213, A342) and glutamic acid (A52). Whereas, on chain B, it consisted of serine (B91) and valine (B342). The CHS, a polyketide synthase, serves as the initial rate-limiting enzyme in the flavonoid biosynthetic pathway.26, 27 According to reports, a decrease in total flavonoid levels was observed when the CHS gene was downregulated through RNA interference in tomato.28

4.4. Chalcone isomerase (CHI), naringenin 3-dioxygenase (F3S), flavanol synthase (FLS), flavonoid 3′-monooxygenase (F3′H), flavanone 4-reductase (FNR) and leucoanthocyanidin dioxygenase (LDOX) in G. sylvestre

Another key rate-limiting enzyme in the flavonoid biosynthesis pathway is CHI.29 The CHI plays a pivotal role by facilitating the intramolecular cyclization of chalcones, leading to the formation of heterocyclic ring C. This process results in the production of various types of flavanones within the cytoplasm.30 The chalcone, the intermediate product gets converted to the stable product, the THC 2′-glucoside [isosalipurposide (ISP)]. This conversion is mediated under the action of chalcone 2′-glucosyltransferase (CH2′GT) in plant vacuoles. Chalcone can also undergo spontaneous isomerization under the influence of CHI resulting in the formation of colourless naringenin.31 In plants, according to the substrate utilized, the CHIs can be classified into two types.29 Type I CHIs, which are found in a wide range of vascular plants, are responsible for transforming THC into naringenin.32 In contrast, Type II CHIs, predominantly present in leguminous plants, can utilize either THC or isoliquiritigenin to produce naringenin and liquiritigenin. Flavonoid content has been observed to have a positive correlation with the expression of CHI in A. thaliana,33 Dracaena cambodiana and tobacco.32 FLS facilitates the oxidation of dihydroflavonols to flavonols creating a double bond between C-2 and C-3 of the C-ring. Its functionality relies on cofactors such as 2-oxoglutarate, ferrous iron (II), and ascorbate.34

FLS transforms dihydroflavonols, including dihydrokaempferol (DHK) into kaempferol, dihydroquercetin (DHQ) into quercetin, dihydrotricetin (DHT) into tricetin and dihydromyricetin (DHM) into myricetin leading to the synthesis of these flavonols. The activity of F3′5′H can lead to the production of myricetin from either kaempferol or quercetin. Flavanone 4-reductase (FNR) catalyzes the enzymatic conversion of flavanones such as naringenin, hesperetin and eriodictyol into flavan-4-ols, specifically apiforol, naringin, neohesperidin and luteoforol which then undergo further polymerization to produce phlobaphenes.35 FNR is a NADPH-dependent reductase and drives the substitution of an oxygen with a hydroxyl group at position C-4 of ring C.

Dihydroflavonol-4-reductase (DFR), an NADPH-dependent reductase is a key player in the flavonoid metabolism, particularly in the anthocyanidin and proanthocyanidin biosynthesis pathways. Its function involves adding a hydroxyl group to position C-4 of ring C.36 DFR is the enzyme responsible for catalyzing the reduction of dihydroflavonols, specifically DHK, DHQ and DHM, leading to the creation of their respective leucoanthocyanidins, also known as flavan-3,4-ols or flavan-diols, including leucopelargonidin, leucocyanidin, and leucodelphinidin.37 Leucoanthocyanidin plays a pivotal role as an intermediate product in the flavonoid pathway, serving as the direct synthetic precursor for both anthocyanidin and proanthocyanidin. The leucoanthocyanidin dioxygenase (LDOX), a type of anthocyanidin synthase (ANS) catalyses these colourless leucoanthocyanidins (leucopelargonidin, leucocyanidin and leucodelphinidin) into the corresponding anthocyanidins (the coloured pelargonidin, cyanidin and delphinidin). The overexpression of these ANS genes has led to an increase in the levels of anthocyanins in the strawberry fruit, resulting in higher concentrations of these vibrant compounds.38

5. Conclusion

Gymnema sylvestre, a non-model plant is primarily recognized for its gymnemic acids; however, it also contains flavonoids. Flavonoids gives a wide variety of colours apart from their role in plant development and defence system. Flavonoids also serve as signaling molecules, attracting insects for pollination and playing a role in auxin metabolism. For the first time in G. sylvestre; an important medicinal plant, a set of 13 major genes are identified and presented in the form of a putative biosynthetic pathway of flavonoids through transcriptome approach. Gene sequences of PAL (OQ190562), 4CL (OQ190563), C4H (MT900543), HST (MT900547), coumaroyl quinate (MT889455), CCOAOMT (MT900548), CHS (MT900539), CHI (MT900540), F3H (MT900541), FLS (MT900542), F3′H (MT900544), C12RT1 (MT900546) and LDOX (MT900545) were analysed. This study primarily emphasised to enhance comprehension of flavonoid biosynthesis at the transcriptome level. It focused on unraveling the processes responsible for the production of flavonoids, a significant group of secondary metabolites, both in the broader context of plants and more specifically in G. sylvestre.

Funding source

This research was financially supported by the FAP Scheme, Gujarat State Biotechnology Mission (GSBTM), Government of Gujarat, Gandhinagar, Gujarat, India. The funds were utilized for purchase of consumables including chemicals and glassware and outsourcing services for sequencing including bioinformatics analysis required in this study.

Research involving Human Participants and/or Animals

There was no involvement of Human Participants and/or Animals in this study.

Author contributions

Kuldeepsingh A. Kalariya: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ravina R. Mevada: Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Manish Das: Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors are grateful to the Indian Council of Agricultural Research, New Delhi and the Director, ICAR-DMAPR, Anand for providing all basic facilities and the Gujarat State Biotechnology Mission, Government of Gujarat, India for funding assistance. We are also thankful to the germplasm collector Dr. P Manivel, Principal Scientist, Plant Breeding, all curators and the Farm Section, ICAR-DMAPR, Anand for maintaining the germplasm used in this study.

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