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 .

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.

Camelina sativa is a promising oilseed and industrial crop that benefits sustainable food, feed and fuel industries. Early flowering is critical for local adaptation as well as maximizing yield in Camelina sativa. Even though the preliminary data indicated wide variation in flowering time in the spring camelina germplasm, our understanding of underlying genes and their roles in regulating flower development is still limited. The current study combined genotypic data and flowering time from the spring panel, followed by genome-wide association study (GWAS) and whole-genome prediction to identify significant trait-associated markers and evaluate the predictive capability of the entire marker set. The analysis of phenotypic data showed significant genotypic and environmental effects on flowering time. A high heritability of 0.893 in flowering time suggests effectiveness of breeding early flowering camelina varieties. The GWAS analysis identified 20 significant trait-associated single nucleotide polymorphisms (SNPs) that colocalized within/or near a variety of transcription factors (e.g. SUPPRESSOR of PHYA-1/SPA1, BES1-INTERACTING MYC-LIKE 1/BIM1) or protein families containing specific functional domains (e.g. CCCH zinc finger protein family and B3-DNA binding domain containing protein family). These transcription factors were known to interact with key regulatory genes in the four major pathways (i.e. photoperiod, autonomous, vernalization and gibberellic acid pathways) to cooperatively regulate floral transition in arabidopsis. Whole-genome prediction showed a low-to-moderate predictive ability (0.559) to improve early flowering trait in camelina. This study is the first step for future in-depth exploration and genetic improvements of flower development and timing in camelina for breeding.

This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6) and was originally published as part of Appendix S1 of "Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes" Marc T. J. Johnson et al. PLOS ONE, November 21, 2012. https://doi.org/10.1371/journal.pone.0050226

Bioengineered lines of the low-input oil seed crop Camelina sativa (Camelina), augmented to accumulate the monoterpene (4S)-limonene, the sesquiterpene (+)-δ-cadinene, or the sesquiterpene (+)-5-epi-aristolochene in seed, were evaluated for two growing seasons under field conditions to determine performance of the introduced traits in an agricultural setup including the effects on overall plant fitness, and total seed yield. Field-grown Camelina plants were further compared to greenhouse-grown plants to evaluate commonalities and differences resulting from cultivation under either controlled or agriculturally relevant growth conditions. Morphological appearance and plant height differed marginally between transgenic and wild-type plants under both greenhouse and field conditions, indicating low impact of the terpene production traits toward overall plant fitness. Total seed yield, however, was independent of the growth conditions and was reduced in Camelina lines producing (4S)-limonene by 48% on average and by 30% on average for (+)-5-epi-aristolochene producing lines. Conversely, (+)-δ-cadinene producing Camelina seed yields remained wild-type equivalent. Additional investigations included seed terpene accumulation, seed oil amount, and seed fatty acid composition. Terpene accumulation was reduced up to 22% for field-grown plants as compared to greenhouse-grown plants. Seed oil amounts were similar under greenhouse and field conditions but were consistently lowered by 2–5% for terpene producing lines. Similarly, seed oil composition remained stable under both field and greenhouse conditions, but generally favored a more energy dense phenotype in terpene producing Camelina lines. Lastly, outcrossing of transgenic traits to adjacent wild-type Camelina plants was observed under field but not greenhouse conditions.

BackgroundThe continued emergence of side-effects caused by synthetic drugs underscores the need for plant-based drugs in human medicine. Medicinal rhizomatous crops are a “goldmine for modern drugs”, and include such species as Gloriosa superba L. and Colchicum autumnale L., the producers of colchicine, a plant-based medicine. The natural isomer of bioactive colchicine is used to effectively treat major diseases such as cancer, cardiovascular disease, and gout. The medicinal properties of colchicine are well characterized, however, almost nothing is known about its biosynthesis. The paucity of information on the colchicine biosynthetic pathway is a significant barrier to biomanufacturing of this biomedicine. A comparative transcriptome study of G. superba and C. autumnale serves as a sequence resource to aid with identification of this biomedicine pathway and rhizome development genes for synthetic biotechnology toolbox, which will enable improved colchicine biomanufacturing.ResultTranscriptomes of two colchicine synthesizing monocots G. superba and C. autumnale were interrogated to identify putative cDNAs encoding enzymes and transcription factors involved in the colchicine biosynthetic pathway and rhizome development. Mining of the transcriptomes using Blast2GO led to the identification from G. superba and C. autumnale, respectively, of 20 and 29 candidate colchicine biosynthetic genes N-methyltransferases, 3-O-methyltransferases, cytochrome P450s, a class that could catalyze several steps in the pathway, and N-acetyltransferases. Similarly, 19 and 15 candidate rhizome developmental genes, which belongs to several classes including GIGANTEA, CONSTANS, Phytochrome B, Sucrose Synthase), Flowering Locus T, and REVOLUTA. Likewise, about 16 and 12 transcription factors involved in regulating rhizome development and secondary metabolic pathways in rhizomes such as MADS-box, AP2-EREBP, bHLH, MYB, NAC, and WRKY were also found in G. superba and C. autumnale, respectively.ConclusionThe predicted genes in G. superba and C. autumnale encode colchicine pathway enzymes that provide fundamental information for plant-based biomedicine engineering in biorhizomes and microorganisms, a potentially important area of synthetic biotechnology. Additionally, increasing our understanding of rhizome functional genomics will lead to improved colchicine biomanufacturing, and generate important knowledge that can be applied to many other medicinal plant species, allowing for the engineered production of additional biomedicines in medicinal rhizomes.

Naturally occurring opiates, such as morphine and thebaine, are produced in poppy species. Thebaine is converted into painkillers and opiate addiction treatments, the latter requiring a chemical reaction called N -demethylation. Current opiate N -demethylation utilizes noxious reagents, resulting in copious amounts of harmful waste. One way to make opiate production more sustainable is to use enzymes rather than chemicals. Microorganisms provide a rich source of enzymes useful for metabolizing unique compounds in their environment. Therefore, an opium-processing waste stream was probed to identify an organism capable of catalysing opiate N -demethylation. A sludge sample was grown on minimal medium containing thebaine as the sole carbon source, to identify a biocatalyst. This led to the discovery of Thebainfresser, a Methylobacterium that metabolizes opiates by removing the N -methyl group. N -demethylation was induced following growth in minimal medium, a characteristic which led to discovery of the underlying gene MND (morphinan N -demethylase). The enzyme MND was found to be robust and versatile, N -demethylating structurally diverse substrates at varying temperatures and pH levels. In addition, MND tolerated selected organic solvents and maintained activity when immobilized. These properties make it an attractive candidate for further development for pharmaceutical manufacture.

There is a need to explore renewable alternatives (e.g., biofuels) that can produce energy sources to help reduce the reliance on fossil oils. In addition, the consumption of fossil oils adversely affects the environment and human health via the generation of waste water, greenhouse gases, and waste solids. Camelina sativa, originated from southeastern Europe and southwestern Asia, is being re-embraced as an industrial oilseed crop due to its high seed oil content (36-47%) and high unsaturated fatty acid composition (>90%), which are suitable for jet fuel, biodiesel, high-value lubricants and animal feed. C. sativa's agronomic advantages include short time to maturation, low water and nutrient requirements, adaptability to adverse environmental conditions and resistance to common pests and pathogens. These characteristics make it an ideal crop for sustainable agricultural systems and regions of marginal land. However, the lack of genetic and genomic resources has slowed the enhancement of this emerging oilseed crop and exploration of its full agronomic and breeding potential. Here, a core of 213 spring C. sativa accessions was collected and genotyped. The genotypic data was used to characterize genetic diversity and population structure to infer how natural selection and plant breeding may have affected the formation and differentiation within the C. sativa natural populations, and how the genetic diversity of this species can be used in future breeding efforts. A total of 6,192 high-quality single nucleotide polymorphisms (SNPs) were identified using genotyping-by-sequencing (GBS) technology. The average polymorphism information content (PIC) value of 0.29 indicate moderate genetic diversity for the C. sativa spring panel evaluated in this report. Population structure and principal coordinates analyses (PCoA) based on SNPs revealed two distinct subpopulations. Sub-population 1 (POP1) contains accessions that mainly originated from Germany while the majority of POP2 accessions (>75%) were collected from Eastern Europe. Analysis of molecular variance (AMOVA) identified 4% variance among and 96% variance within subpopulations, indicating a high gene exchange (or low genetic differentiation) between the two subpopulations. These findings provide important information for future allele/gene identification using genome-wide association studies (GWAS) and marker-assisted selection (MAS) to enhance genetic gain in C. sativa breeding programs.

1Department of Genetics, University of Georgia, Athens, GA 30602, USA 2Department of Plant Biology, University of Georgia, Athens, GA 30602, USA 3Donald Danforth Plant Science Center, St. Louis, MO 63132, USA 4Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK 5Athens, OH 45701, USA 6Department of Biology, University of Washington, Seattle, WA 98195, USA 7Department of Biology, Pennsylvania State University, University Park, PA 16803, USA 8Department of Biological Science, University of Alberta, Edmonton, AB, T6G 2E9, Canada 9BGI-Shenzhen, Bei shan Industrial Zone, Yantian District, Shenzhen 518083, China 10Department of Medicine, University of Alberta, Edmonton, AB, T6G 2E1, Canada

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.

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.

Hussein Abdel-Haleem1* (Hussein Abdel-Haleem@usda.gov), Zinan (Lily) Luo1, Aaron E. Szczepanek1, Matthew Conley1, Hao Zhang2, Yufeng Ge2, Daniel Schachtman2, Megan M. Augustin3, Jordan R. Brock3, Noah Fahlgren3, Toni Kutchan3, Sheeja George4, Russ Gesch5, and John M. Dyer1 1USDA-ARS, US Arid-Land Agricultural Research Center, Maricopa, AZ; 2University of Nebraska-Lincoln, Lincoln, NE; 3Donald Danforth Plant Science Center, St. Louis, MO; 4University of Florida, Quincy, FL; and 5USDA-ARS, North Central Soil Conservation Research Laboratory, Morris, MN

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 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.

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, and mass spectrometry.