Evaluation of reference genes for qRT-PCR studies in the colchicine producing Gloriosa superba L.

The flame lily, Gloriosa superba L., is one of the two primary sources of the anti-inflammatory drug, colchicine. Previous studies have shown that a higher level of colchicine production occurs in the rhizomes than in leaves and roots. Earlier precursor feeding and transcriptome analysis of G. superba have provided a putative pathway and candidate genes involved in colchicine biosynthesis. Comparative analysis of expression levels of candidate pathway genes in different tissues of G. superba using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) can reveal highly expressed genes in the rhizome compared to other tissues which could suggest roles of the gene products in colchicine biosynthesis. Normalization is an important step in effectively analyzing differential gene expression by qRT-PCR with broader applications. The current study selected candidate reference genes from the transcriptome datasets and analyzed them to determine the most stable genes for normalization of colchicine biosynthesis-related genes. Using RefFinder, one stable reference gene, UBC22, was selected to normalize gene expression levels of candidate methyltransferase (MT) genes in the leaves, roots, and rhizomes of G. superba. With UBC22 as reference gene, the methyltransferases, GsOMT1, GsOMT3, and GsOMT4 showed significantly higher expression levels in the rhizome of G. superba, while MT31794 was more highly expressed in the roots. In conclusion, the current results showed a viable reference gene expression analysis system that could help elucidate colchicine biosynthesis and its exploitation for increased production of the drug in G. superba. Supplementary Information: The online version contains supplementary material available at 10.1007/s11816-023-00840-x.

Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis.

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope.

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

Plant metabolism, the diverse chemistry set of the future.

New technologies are redefining how plant biology will meet societal challenges in health, nutrition, agriculture, and energy. Rapid and inexpensive genome and transcriptome sequencing is being exploited to discover biochemical pathways that provide tools needed for synthetic biology in both plant and microbial systems. Metabolite detection at the cellular and subcellular levels is complementing gene sequencing for pathway discovery and metabolic engineering. The crafting of plant and microbial metabolism for the synthetic biology platforms of tomorrow will require precise gene editing and delivery of entire complex pathways. Plants sustain life and are key to discovery and development of new medicines and agricultural resources; increased research and training in plant science will accelerate efforts to harness the chemical wealth of the plant kingdom.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery.

Amaryllidaceae alkaloids are an example of the vast diversity of secondary metabolites with great therapeutic promise. The identification of novel compounds in this group with over 300 known structures continues to be an area of active study. The recent identification of norbelladine 4'-O-methyltransferase (N4OMT), an Amaryllidaceae alkaloid biosynthetic enzyme, and the assembly of transcriptomes for Narcissus sp. aff. pseudonarcissus and Lycoris aurea highlight the potential for discovery of Amaryllidaceae alkaloid biosynthetic genes with new technologies. Recent technical advances of interest include those in enzymology, next generation sequencing, genetic modification, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS).

CYP96T1 of Narcissus sp. aff. pseudonarcissus Catalyzes Formation of the Para-Para' C-C Phenol Couple in the Amaryllidaceae Alkaloids.

The Amaryllidaceae alkaloids are a family of amino acid derived alkaloids with many biological activities; examples include haemanthamine, haemanthidine, galanthamine, lycorine, and maritidine. Central to the biosynthesis of the majority of these alkaloids is a C-C phenol-coupling reaction that can have para-para', para-ortho', or ortho-para' regiospecificity. Through comparative transcriptomics of Narcissus sp. aff. pseudonarcissus, Galanthus sp., and Galanthus elwesii we have identified a para-para' C-C phenol coupling cytochrome P450, CYP96T1, capable of forming the products (10bR,4aS)-noroxomaritidine and (10bS,4aR)-noroxomaritidine from 4'-O-methylnorbelladine. CYP96T1 was also shown to catalyzed formation of the para-ortho' phenol coupled product, N-demethylnarwedine, as less than 1% of the total product. CYP96T1 co-expresses with the previously characterized norbelladine 4'-O-methyltransferase. The discovery of CYP96T1 is of special interest because it catalyzes the first major branch in Amaryllidaceae alkaloid biosynthesis. CYP96T1 is also the first phenol-coupling enzyme characterized from a monocot.

Production of mono- and sesquiterpenes in Camelina sativa oilseed.

MAIN CONCLUSION: Camelina was bioengineered to accumulate (4 S )-limonene and (+)-δ-cadinene in seed. Plastidic localization of the recombinant enzymes resulted in higher yields than cytosolic localization. Overexpressing 1-deoxy- d -xylulose-5-phosphate synthase ( DXS ) further increased terpene accumulation. Many plant-derived compounds of high value for industrial or pharmaceutical applications originate from plant species that are not amenable to cultivation. Biotechnological production in low-input organisms is an attractive alternative. Several microbes are well established as biotechnological production platforms; however, their growth requires fermentation units, energy input, and nutrients. Plant-based production systems potentially allow the generation of high-value compounds on arable land with minimal input. Here we explore whether Camelina sativa (camelina), an emerging low-input non-foodstuff Brassicaceae oilseed crop grown on marginal lands or as a rotation crop on fallow land, can successfully be refactored to produce and store novel compounds in seed. As proof-of-concept, we use the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon (+)-δ-cadinene, which have potential biofuel and industrial solvent applications. Post-translational translocation of the recombinant enzymes to the plastid with concurrent overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) resulted in the accumulation of (4S)-limonene and (+)-δ-cadinene up to 7 mg g(-1) seed and 5 mg g(-1) seed, respectively. This study presents the framework for rapid engineering of camelina oilseed production platforms for terpene-based high-value compounds.

Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

Phylotranscriptomic analysis of the origin and early diversification of land plants.

Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Data access for the 1,000 Plants (1KP) project.

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

Anthraquinone Content in Noni (Morinda citrifolia L.).

Noni has been used in traditional medicine and as food for thousands of years. While the fruits serve as food and internal medicine, leaves were traditionally used only topically. In recent years, concern regarding the possible content of anthraquinones in noni has led to scrutiny by the European Food Safety Authority. Little research existed on the content of anthraquinones in different noni preparations, with no information about the potential effect of harvest and preparation methods. Our research focused on lucidin, alizarin, and rubiadin, the most important anthraquinones from a health perspective. We found that the production process (fermentation/juice production versus drying/lyophilization) has no effect on the anthraquinone content. The source product, however, does have implications: noni fruit puree from which seeds had been removed as well as consumer products produced from such puree had no detectable amounts of any anthraquinones. Products that did contain seed or leaf material in all cases did contain partly significant amounts of anthraquinones. To alleviate safety concerns, we suggest that noni products, whether fermented or unfermented juice or powder, should be derived only from fully ripe noni fruits, and that any seed material needs to be removed during the production process.

Cyclotide discovery in Gentianales revisited--identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate.

The bicyclic diterpene (-)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (-)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 [M+H](-)). The enzymatic reaction was also carried out in the presence of 60% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases.

Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes.

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥ 1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation.

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

A genome triplication associated with early diversification of the core eudicots.

BACKGROUND: Although it is agreed that a major polyploidy event, gamma, occurred within the eudicots, the phylogenetic placement of the event remains unclear. RESULTS: To determine when this polyploidization occurred relative to speciation events in angiosperm history, we employed a phylogenomic approach to investigate the timing of gene set duplications located on syntenic gamma blocks. We populated 769 putative gene families with large sets of homologs obtained from public transcriptomes of basal angiosperms, magnoliids, asterids, and more than 91.8 gigabases of new next-generation transcriptome sequences of non-grass monocots and basal eudicots. The overwhelming majority (95%) of well-resolved gamma duplications was placed before the separation of rosids and asterids and after the split of monocots and eudicots, providing strong evidence that the gamma polyploidy event occurred early in eudicot evolution. Further, the majority of gene duplications was placed after the divergence of the Ranunculales and core eudicots, indicating that the gamma appears to be restricted to core eudicots. Molecular dating estimates indicate that the duplication events were intensely concentrated around 117 million years ago. CONCLUSIONS: The rapid radiation of core eudicot lineages that gave rise to nearly 75% of angiosperm species appears to have occurred coincidentally or shortly following the gamma triplication event. Reconciliation of gene trees with a species phylogeny can elucidate the timing of major events in genome evolution, even when genome sequences are only available for a subset of species represented in the gene trees. Comprehensive transcriptome datasets are valuable complements to genome sequences for high-resolution phylogenomic analysis.