The costunolide biosynthesis enzymes of Artemisia glabella Kar. et Kir.: Determination of the nucleotide sequences of the mRNA

Shaikenova Zh. S.; Chervyakova V.; Zakarya K. D.; Adekenov S.

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The costunolide biosynthesis enzymes of Artemisia glabella Kar. et Kir.: Determination of the nucleotide sequences of the mRNA Creative Commons

Adekenova Gulimzhan S.,

Shaikenova Zh. S.,

Chervyakova V.,

Zakarya K. D.,

Adekenov S.

International Journal of Plant Physiology and Biochemistry, Journal Year: 2021, Volume and Issue: Vol.13(1), P. 9 - 18

Published: April 30, 2021

Abstract

Artemisia glabella Kar. et Kir. is a source of sesquiterpene lactone arglabin, which has antitumor, radiosensitizing and immunomodulatory activity. Studying the biosynthesis of arglabin and its derivatives will allow us to develop the biotechnological basis for its production, thereby increasing its availability. The precursor to most sesquiterpene lactones is costunolide. The purpose of these studies was to detect and determine the nucleotide sequences of mRNA enzymes germacrene A synthase (GAS, EC 4.2.3.23), germacrene A oxidase (GAO, EC 1.14.14.95) and costunolide synthase (COS, EC 1.14.13.120), involved in the biosynthesis of costunolide in A. glabella. As a result of studies, mRNA was isolated from various forms (intact plant, regenerant plant, callus) of A. glabella. Using specific primers, mRNA fragments of genes encoding sesquiterpene lactones biosynthesis enzymes were amplified and their nucleotide sequences were determined. A comparative analysis of the obtained sequences showed their high (>90%) identity with the genes of GAS, GAO and COS of other representatives of the family Asteraceae. It was revealed that enzymes are expressed both in an intact plant and in calluses and regenerant plants obtained in vitro.

Keywords

Artemisia glabella Kar. et Kir., costunolide synthase., mRNA, germacrene A synthase, germacrene A oxidase, cDNA, sequencing

INTRODUCTION

Sesquiterpenoids of the guaiane series belong to fairly widespread substances of the plant kingdom. A particularly large number of guaiane compounds are included in the group of sesquiterpene lactones called guaianolides. α-Methylene guaianolides have a bitter taste, are toxic, and exhibit cytotoxic and antitumor properties. Their molecules, as a rule, are polyfunctional and many of them are characterized by the presence of exocyclic unsaturation (Fischer et al., 1979; Frederick, 1982).

Sesquiterpene lactones are derivatives of isopentenyl diphosphate (IPP), which can be synthesized in two

different ways: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In the MEP pathway, glyceraldehyde-3-phosphate and pyruvic acid are converted to IPP and DMAPP by seven enzymes. In the MVA pathway, acetyl-CoA is converted to IPP through six steps followed by IPP isomerase (IPPI). Two IPPs and one DMAPP are then converted to farnesyl diphosphate (1) (FPP), the precursor of all sesquiterpenes, by farnesyl diphosphate synthase (FPS). FPP (1) can be converted to sesquiterpenes using sesquiterpene synthases FPP (1).

Sesquiterpene lactones are formed in the plant organism by oxidation of the methyl part of the isopropyl group attached to the main carbon skeleton. Cyclization of farnesyldiphosphate (1) gives (+)- germacrene A (2). Oxidation of the isopropenyl side chain by (+)- germacrene A-hydroxylase to primary alcohol and further oxidation by NAD (P)+- dependent dehydrogenases gives germacrene acid (3). This is followed by hydroxylation at the C-6 position and subsequent lactonization leads to (+)- costunolide (4). It is assumed that the second stage of the cyclization of germacranolides to the guaianolide skeleton proceeds through epoxidation or hydroxylation of the costunolide skeleton (Ma et al., 2019).

As a result of research on the homology of nucleotide sequences with known sesquiterpene monooxygenases, six promising cytochrome P450 contigs (actin, GAS, GAO, COS, parthenolide synthase and costunolide 3β-hydroxylase) were identified and selected for functional characterization. A new cytochrome P450, cauniolide synthase, which catalyzes the formation of guaianolide cauniolide (5) from the germacranolide substrate costunolide (4), has been characterized. Unlike most cytochromes P450s, cauniolide synthase has a unique mechanism of action, combining stereoselective hydroxylation of costunolide (4) at the C-3 position with elimination, cyclization, and regioselective deprotonation (Liu et al., 2018).

One of the promising guaianolides is arglabin (6), isolated for the first time from Artemisia glabella Kar. et Kir., an endemic species of wormwood (Artemisia L.), which grows exclusively in Central Kazakhstan. A. glabella is ? source of a number of biologically active compounds such as sesquiterpene lactones and essential oils (Adekenov et al., 1982; Adekenov et al., 1995). Arglabin (6) is the active substance of the anticancer drug “Arglabin”, which was developed at the International Research and Production Holding “Phytochemistry” and is produced on an industrial scale by the Karaganda Pharmaceutical Plant (Karaganda, Republic of Kazakhstan) (Adekenov, 2001, 2015).

Arglabin (6) is found in all organs of A. glabella and throughout the growing season (Adekenov et al., 1995). Its quantitative accumulation is observed during the budding period in leaves (1.90%) and buds (1.56%) (Mantler et al., 2020). However, the biosynthesis stages of the arglabin molecule in a plant organism at the genetic level are studied insufficiently.

Promising approach to obtain arglabin (6) can be the reconstruction of the biosynthetic pathway in a heterologous system. However, despite the widespread medical use of arglabin (6), the biosynthetic pathway in plants remains unexplored. Adekenov and Bouwmeester (2015) described a possible pathway for arglabin biosynthesis in A. glabella: farnesyldiphosphate (1), germacrene A (2), germacrene A-ol (7), germacrene A-on (8), germacrene acid A (3), costunolide (4), cauniolide (5), and arglabin (6) (Figure 1).

The formation of germacrene A (2) as a result of the cyclization of farnesyl diphosphate (1) catalyzed by germacrene A synthase (GAS) is a generally recognized step in the biosynthesis of sesquiterpenes (Xu and Dickschat, 2020). GAS was isolated and characterized from a number of plants of the family Asteraceae (Bouwmeester et al., 2002; Majdi et al., 2011; Menin et al., 2012; Pazouki et al., 2015; Nguyen et al., 2016). The next step in biosynthesis is the sequential oxidation of germacrene A (2) by germacrene A oxidase (GAO) and costunolide synthase (COS). As a result, germacrene acid (3) and costunolide (4) are formed, respectively (Nguyen et al., 2019; de Kraker et al., 2002; Ikezawa et al., 2011).

In previous studies, a number of genes encoding sesquiterpene enzymes that control key steps in secondary metabolic pathways have been characterized from a number of herbal plant species. However, the genes responsible for enzymatic processes in guaianolide biosynthesis have not been identified. Therefore, the isolation and characterization of genes involved in the biosynthesis of arglabin (6) in A. glabella is a promising direction of research for the reconstruction of the process in a heterologous system. The main objectives of this study were the isolation of genes encoding the GAS, GA? and C?S in A.glabella and the determination of the nucleotide sequences of mRNA, as well as the assessment of the expression of these genes at the level of transcription in regenerated plants and callus tissues.

MATERIALS AND METHODS

Plant

The intact plant of A. glabella Kar. et Kir. in the budding phase under ex situ conditions, the regenerant plant and callus tissues obtained in vitro culture in the biotechnology laboratory of JSC “International Research and Production Holding “Phytochemistry” were used. Leaves and flower buds were collected and immediately frozen in liquid nitrogen and stored at -80°C (three biological replicates for each tissue were used).

RNA isolation: cDNA synthesis

For total RNA extraction, fresh or freezed plant material (about 100 mg) immediately transferred to 200 μl Tryzol reagent in 1.5 ml microcentrifuge tube. Cells were disrupted mechanically with a pestle in the presence of carborundum. After cell disruption, an additional 800 μl Tryzol reagent was added. All subsequent steps were carried out as described in the manual.

mRNA was isolated from total RNA using the Dynabeads® mRNA DIRECT™ Purification Kit (Invitrogen, USA) according to the manufacturer's protocol.

cDNA synthesis was performed using the Maxima H Minus Double-Stranded cDNA Synthesis Kit (Thermo Scientific, USA), according to the manufacturer's protocol, with oligo (dT) primer.

Amplification and sequencing of fragments of targeted genes

Amplification of cDNA fragments of targeted genes was carried out using specific primers designed as part of these studies. A 50 μl reaction mixture contained: 5 μl 10×PCR buffer (Silex, Russia), 1 μl 10 mM dNTPs (NEB), 0.1 μl cDNA (100 ng/μl), 1 μl of each primer (20 pM/μl), 0.25 μl Taq DNA polymerase (1.25 units, Sileks). Amplification was carried out under the following conditions: at 94°C 5 min; then 30 cycles at 94°C 1 min; at 50°C 1 min; at 68°C 2 min; final elongation at 68°C 7 min.

PCR products were cloned in pJet1.2 vector using the Clone JET PCR Cloning Kit (Thermo Scientific, USA) according to the manufacturer's instructions. Competent cells of Escherichia coli TOP10 were transformed with ligation mix. Presence and size of insertions in plasmid vector was determined by PCR analysis of E. coli TOP10 colonies according to the manufacturer's instructions for CloneJET PCR Cloning Kit (Thermo Scientific). Plasmid DNA was isolated from E. coli TOP10 using the GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol.

Nucleotide sequences of targeted genes were determined using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Austin, TX), on the DNA sequencer Genetic Analyser 3130xl, Applied Biosystems, Hitachi, Japan. Nucleotide and their relevant amino acid sequences were analyzed with the help of the software Vector NTI 10.0.1, Invitrogen. The GAS/GAO/COS nucleotides from the GenBank databases were used for comparison.

Phylogenetic analyses

The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

RESULTS AND DISCUSSION

Design of primers

The design of primers for amplification of target genes was carried out based on the analysis of the nucleotide and amino acid sequences of GAS, GAO and COS in members of the Asteraceae family presented in the GenBank of The National Center for Biotechnology Information, U.S. National Library of Medicine.

Amino acid sequences of GAS, GAO, COS are presented in GenBank for 17, 11 and 8 species of the Asteraceae family, respectively. Two forms of GAS were found: short ~ 560 amino acids (a.a.) and long ~ 580 a.a. The average the length of the amino acid sequences of GAO is 488 a.a. and COS is 494 a.a.

Conserved short peptides were identified based on the multiple alignments of the amino acid sequences of GAS, GAO, COS (Figure 2). These peptides became targets for the design of specific primers (Table 1).

Phylogenetic analysis

Pairwise protein sequences comparison of putative AgGAS, AgGAO and AgCOS with all known enzymes shows wide range of homology.

In silico analysis revealed that putative AgGAS has about 40 to 90% homology at protein level with annotated germacrene A synthases. It has highest identity (90%) with Tanacetum parthenium germacrene synthase (TpGAS) and lowest identity (40%) with Solidago canadensis germacrene synthases (ScGAS) (Figure 4A). Neighbour joining algorithm generated phylogenetic tree has combined AgGAS, AaGAS and TpGAS into one evolutionary lineage (Figure 4B). Tanacetum and Artemisia are members of the taxonomic tribe Anthemideae.

Phylogenetic analysis of AgGAO and AgCOS showed high homology with the corresponding enzymes of T. parthenium, 97 and 95%, respectively (Figures 5A and 6A). Neighbour joining algorithm generated phylogenetic tree showed separate evolutionary lineage of AgGAO and AaGAO (Figure 5B). However, AgCOS was combined in one lineage with TpCOS (Figure 6B).

Expression of target genes in plant tissues

Expression of AgGAS, AgGAO and AgCOS was evaluated at the transcription stage in regenerant plant and callus tissues obtained in vitro. As a result, we found that mRNA of costunolide (4) biosynthesis enzymes is present both in regenerant plants and in callus tissues (Figure 3B and C). Regenerated plants and callus tissue can also provide an alternative source for arglabin (6) production.

CONCLUSION

Thus, for the first time the nucleotide sequences of mRNA fragments encoding AgGAS, AgGAO and AgCOS were determined. A comparative analysis of the obtained sequences showed their high (> 90%) identity with the annotated GAS, GAO and COS of other members of the family Asteraceae. It was found that AgGAS, AgGAO and AgCOS are expressed both in the intact plant, as well as in calluses and regenerated plants obtained in vitro.

The results obtained provide a basis for further study of the biosynthetic pathway of arglabin (6) in plants. Isolation and characterization of enzymes will make it possible to reconstruct the biosynthesis of arglabin (6) in a heterologous system and to simplify the methods of its preparation for pharmaceutical production.

CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

ACKNOWLEDGEMENTS

This work was carried out according to grant project No. AP05134198 “The study of the biosynthesis of terpenoids in plants and the search for new pharmacologically active bimolecular compounds”, funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan.

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