Understanding AAs metabolism using integrated transcriptomics and metabolomics in Narcissus pseudonarcissus ‘King Alfred’ 

Materials and methods

Plant mate rial and chemicals. Omarnental bulbs of N pseudonarcissus cultivar ‘King Alfred’ were purchased from Fraser’s Thimble farrns (BC, Canada). Bulbs were planted in September in well-drained soil to grow until the flowering stage in the spring.
Different plant tissues such as bulbs, roots, stems, leaves and flowers were harvested in May for metabolic and transcriptomic analyses. HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (https://www.fishersci.com). Standards narciclasine and galantarnine were purchased from Tocris Bioscience (Bristol, U.K.) whereas lycorine and papaverine was purchased from Sigma-Aldrich (ON, Canada).
Alkaloid extraction and HPLC analysis. N pseudonarcissus ‘King Alfred’ plant tissues (2 g/each in triplicate; bulbs, roots, stems, leaves and flowers) were crushed in liquid nitrogen and extracted with methanol (10 ml) for 24 hrs at RT on a shaker (200 rpm). Extracts were centrifuged at 12,000 rpm for 5 minutes to coIlect supematant without debris and left for complete evaporation. The raw alkaloids obtained were further subjected to a modified acid-base extraction method (specific for alkaloids), where they were resuspended in methanol 100% pH-8 (adjusted with NH3) and H2S04 (2% v/v). Organic impurities were removed by washing twice with chloroform. Alkalization was do ne using NH3. The purified alkaloid extracts obtained were dried under N2 gas and finaIly solubilized in 300 ilL methanol. Qualitative analysis of alkaloids was performed on TLC silica gel 60 F254 aluminum sheets 20 x 20 cm, (Merck, Darmstadt, Germany).
Ten III of 100 ppm for each standard (GAL, L YC, NAR) and 15 III of samples were loaded in triplicates on TLC and visualized under 280 nm and 365 nm. For qualitative and quantitative analyses of alkaloids, each sample extract was diluted in 1/1 0 ratio using 1 % ammonium acetate buffer. 15 ul of each sample was injected and analyzed on Shimadzu Prominence-i LC-2030C with diode array detector (PDA). HPLC oyen temperature was set at 40°C. Chromatography assay was performed on Kinetex C18 colurnn (150 x 4.6 mm, 5 Ilm particle size; Phenomenex). Elution was carried out in gradient mode with 1 % ammonium acetate buffer (solvent A) and 100 % acetonitrile (solvent B). InitiaIly 90: 10 gradient ratio of solvent B and solvent A was maintained for 10 min. EventuaIly, gradient was shifted to 69:31 over 5 min, 10:90 over 2 min, and then 90: 1 0 over 3 min. EventuaIly, after 18 min ammonium acetate was reduced to 10% and acetonitrile was increased to 90%, continuing till 30 min. HPLC Chromatograms of aIl standards and plant samples were extracted at 280 nm.
UPLC-QTOF-MS analysis. The UPLC analysis was performed using a Waters Acquit Y Ultra-Performance LC system (Waters), equipped with a binary pump system (Waters). An Acquit Y Ethylene Bridged Hybrid (BEH) C18 colurnn (100 mm_2.1 mm id, 1.7 mm particle size) from Waters was used. The molecules were separated with a mobile phase that consisted of 0.2% acetic acid (solvent A) and 100 % acetonitrile (solvent B).
The flow-rate was 0.2 mL/min and the gradient elution was initial, 2% B ; 0-1 min, 2- 100% B; 1-30 min, isocratic 100% B; 30-33 min, 100-2% B ; 33-33 .5 min, isocratic 2% B; 33-40 min. The MS analyses were carried out on a QTOF Micro mass spectrometer (Waters) equipped with a Z-spray electrospray interface. The analysis was perforrned in both positive and negative mode and the data were acquired through a mass scan from 100 to 1250 m/z without collision. The ionization source parameters were source temperature, 120°C; cone gas flow rate, 50 L/h and desolvation gas flow rate, 350 L/h; desolvation temperature, 200°C. The cone and capillary voltages were respectively set at 30 V and 1150 V for the negative mode and 70 V and 1800 V for the positive mode. Nitrogen (99% purity) was used as nebulizing gas. Data acquisition was carried out with the MassLynx 4.1 software. Masses extraction, deconvolution, isotopes and library search was perforrned using MZMine 2 according to Pluskal et al. (2010) (Pluskal, Castillo et al. 2010).
RNA extraction, next-generation Illumina sequencing, and de novo assembly.Five grams oftriplicate bulbs, stems, roots, leaves or flowers of N pseudonarcissus ‘King Alfred’ were crushed using a pestle and a mortar with liquid nitrogen and transferred to pre-chilled 50 ml tubes to proceed with CTAB (cetrimonium bromide) method for total RNA extraction(Desgagne-Penix, Farrow et al. 2012). After RNA extraction, bulb RNA was selected for Illumina sequencing, and stem, root, leaf and flower RNA were used for RT-qPCR analysis. For transcriptome analysis, integrity of the bulb RNA was checked on a nanodrop and bioanalyzer. Nanodrop quantification yielded 805.68 ng/j.!l of total RNA with the ratio 260 nm/230 nm of2.12 and 260 nm/280 nm of 1.91. Bioanalysis of RNA gave a RNA integrity number (RIN) of 8.3 with 28S/18S of 1.546656 confirrning the RNA quality as pure and intact i.e. not degraded.
The mRNA were converted into cDNA library and sequenced through Illumina HiSeq 2000, PE 100 paired ends, at McGill University and Genome Quebec Innovation Centre (Montreal, Canada). Raw paired reads were trimmed from 3′ -end and Illumina sequencing adapters were removed, maintaining 50 bps of minimum read length to obtain surviving paired reads. Trimming and clipping were done using Trimmomatic surviving pair data were generated, normalization was performed to eliminate redundant reads in datasets without affecting its Kmer content. Final obtained unigenes were functionally annotated usmg trinotate (http://trinotate.github.io/), usmg the Trinit Y normalization utility inspired by the diginorm algorithrn (Brown, Howe et al. 2012). Normalized reads were used to assemble the transcriptome using the Trinit y assembler (Grabherr, Haas et al. 2011). To quantify the gene transcript abundance, the raw RNA-Seq reads were mapped to assembled transcripts applying Bowtie (Langrnead, TrapneIl et al. 2009) using default parameters. The gene transcript abundance was calculated as ‘ fragments per million mapped reads per kilobase’ (FPKM) using the RSEM package(Li and Dewey 2011). The genes with extremely low FPKM values i.e. with a maximum FPKM of less than 1 across two samples, were filtered out before subsequent analysis.
RT -qPCR analysis. Two micrograms of RNA from different tissues (bulb, root, stern, leaf, and flower) of triplicate plants of N pseudonarcissus ‘King Alfred’ were reverse transcribed to form cDNA using oligo dT primers and the Ornniscript Reverse Transcription Kit (Qiagen) according to given manufacturer’s protocol. Gene specific prim ers were designed using Integrated DNA Technology (www.idtdna.com) and Tm Ca1culator, New England Biolabs (trnca1culator.neb.com), to select suitable annealing temperatures. The sequences for aIl of the prim ers used in this study are listed in table C.3 .
SensiFAST SYBER Lo-ROX mix (Bioline) was used to prepare a 20 ~l reaction containing lx SensiF AST SYBER Lo-ROX mix, 200 ~M of each forward and reverse primer and 3 ~l of cDNA sample. Each experiment was performed in triplicate and histone was used as an internaI reference gene. Real-time quantitative PCR was perforrned on CFX Connect Real-Time PCR System (BioRad). PCR conditions for amplification were 95°C for 3 min, 95°C for 10 sec and annealing temperature (varies with gene primers, see Table C.3) for 30 sec for 40 cycles. This was followed by a dissociation step (as provided by software) – 95°C for 10 sec, 65°C for 5 sec and 95°C for 5 sec. The comparative 2-MCt method (Livak and Schrnittgen 2001) was used for relative quantification of the gene expression levels.
Accession numbers. The sequences described in this paper have been deposited III the National Center for Biotechnology Infonnation Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/) under the accession number SRR5788585. Gene transcript sequences were deposited in Genbank with the following accession numbers for nucleotide sequences: tyrosine decarboxylase 1 (MF405171), tyrosine de carboxylase 2 (MF405172),phenylalanine ammonia lyse 1 (MF405173),phenylalanine ammonia lyse 2 (MF405174), cinnamate 4-hydroxylase (MF416091), coumarate 3-hydroxylase (MF416092), 4-coumarate-CoA ligase 1 (MF416093), 4-coumarate-CoA ligase 2 (MF416094), hydroxycinnamoyltransferase (MF416095), norbelladine 4′-0- methyltransferase (MF416096), noroxomaritidine synthase 1 (MF416097), noroxomaritidine synthase 2 (MF416098), noroxomaritidine/norcraugsodine reductase (MF416099) and histone (MF405170).

Acknowledgments

We kindly thank Prof essor Hugo Gennain and Tarun Hotchandani for the revision and helpful comments on a previous version of this manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) award number RGPIN 05294-2014 (Discovery) to LD-P. This work was also supported by the NSERC award number EQPEQ 472990-2015 (Research tools and instruments) for the acquisition of the HPLC- PDA and the qPCR.

Contributions

IDP and AS conceived and designed the experiments. AS performed the experiments. MAM and LT performed LC-MS/MS analysis. VO and GB chemically synthesized the norbelladine and norcraugsodine standards and validate them using NMR analysis. IDP and AS interpreted results, prepared the figures, wrote and reviewed the manuscript.

Abstract

Amaryllidaceae alkaloids (AAs) are a large group ofplant-specialized metabolites displaying an array ofbiological and pharmacological properties. Previous investigatio_ns on AA biosynthesis have revealed that aIl AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. SpecificaIly, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis. With the availability ofwild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes
for a small protein of 19 KDa with an anticipated pl of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PRI OlBet vI proteins and shared 41 % amino acid identity to Papaver sominferum NCS 1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry. Here, we introduce a novel enzyme catalyzing the first committed step of AA biosynthesis and will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.

Introduction

The Amaryllidaceae alkaloids (AAs) are a group of naturally synthesized molecules with more than 500 renowned complex structures (Jin and Xu 2013). They are pharmacologically active compounds that are classified under three different groups ofCC phenol coupling namely para-para ‘, ortho-para ‘ and para-ortho ‘ (Singh and Desgagné-Penix 2014). An outsized variety ofpharmacologically active AAs have been identified with the bioactive properties including the acetylcholine esterase inhibitor galanthamine, anti-tumor activity of lycorine and the cytotoxic haemanthamine (Li, Dai et al. 2012, He, Qu et al. 2015, Hotchandani and Desgagne-Penix 2017). AAs are obtained chiefly from the extracts of plants from Galanthus, Leucojum and Narcissus species, as their complicated structures does not enable cost-effective high-yield organic synthesis (Saliba, Ptak et al. 2015). Though AAs display a large range of pharmaceutical applications, solely galanthamine is accessible in markets as an Alzheimer’ s treatment drug because of its ability to stabilize behavioral symptoms in the course of six months treatment in comparison to chemically synthesized acetylcholine esterase inhibiting drugs, donepezil and rivastigmine (Prvulovic, Hampel et al. 2010).

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Table des matières

ACKNOWLEDGEMENTS
RESUME
ABSTRACT
LIST OF FIGURES
LIST OF ABBREVIATIONS AND ACRONYMS
CHAPTERI
INTRODUCTION
1.1 Specialized Metabolites – A medicinal resource
1.2 Classification of specialized metabolites
1.2.1 Phenolics
1.2.2 Terpenes
1.2.3 Alkaloids
1.3 Transportation and storage of specialized metabolites
1.4 Classification of alkaloids
1.4.1 Purine alkaloids
1.4.2 Tropane alkaloids
1.4.3 Pyrrolizidine alkaloids
1.4.4 Quinolizidine alkaloids
1.4.5 Monoterpene Indole alkaloids
1.4.6 Isoquinoline alkaloids
1.4.6.1 Benzylisoquinoline alkaloids
1.4.6.2 Amaryllidaceae alkaloids
1.4.6.2.1 Biological roI es of AAs
1.4.6.2.2 Amaryllidaceae alkaloids biosynthesis
1.5 Discovery ofunknown genes using Systems Biology
1.6 Importance, hypotheses, and objectives
1.6.1 Objective 1: Understanding AAs metabolism using integrated transcriptomics and metabolomics in Narcissus pseudonarcissus ‘King Alfred’
1.6.2 Objective II: Cloning and characterization of Norbelladine synthase, a novel gene involved in norbelladine synthesis in Narcissus pseudonarcissus ‘King Alfred’
CHAPTERII
TRANSCRIPTOME AND METABOLOME PROFILING OF NARCISSUS PSEUDONARCISSUS ‘KING ALFRED’ REVEAL COMPONENTS OF AMARYLLIDACEAE ALKALOID METABOLISM 
2.1 Contribution
2.2 Abstract
2.3 Introduction
2.4 Results
2.5 Discussion
2.6 Material and methods
2.7 Acknowledgements
2.8 References
CHAPTERIII
CLONING AND CHARACTERIZATION OF NORBELLADINE SYNTHASE CATALYZING THE FIRST COMMITTED REACTION AMARYLLIDCEAE ALKALOID BIOSYNTHESIS
3.1 Contribution
3.2 Abstract
3.3 Introduction
3.4 Materials and methods
3.5 Results
3.6 Discussion
3.7 Acknowledgements
3.8 References
CHAPTERIV
CONCLUSIONS
4.1 Conclusion
4.2 FuturePerspectives
4.3 Final conclusion
REFERENCES
ANNEXA
BIOSYNTHESIS OF AMARYLLIDACEAE ALKALOIDS
ANNEXB
BIOSYNTHESIS OF AMARYLLIDACEAE ALKALOIDS: A BIOCHEMICAL OUTLOOK
ANNEXC
SUPPLEMENTARY DATA OS CHAPTER II
ANNEXD
SUPPLEMENTARY DATA OF CHAPTER IlL

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