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.
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.
, 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.
'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
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
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
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
breeding programs.
Summary
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.
Significance Statement
The first four enzymes involved in the biosynthesis of the steroid alkaloid verazine, a predicted precursor to the antineoplastic cyclopamine in Veratrum californicum, were discovered: cholesterol 22‐hydroxylase, 22‐hydroxycholesterol 26‐hydroxylase/oxidase, 22‐hydroxycholesterol‐26‐al transaminase, and 22‐hydroxy‐26‐aminocholesterol 22‐oxidase. The pathway to verazine has been refactored in Spodoptera frugiperda Sf9 cells.
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.
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.
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•MUT9-LIKE KINASE mutant quantitative proteome and phosphoproteome measured.•Changes to proteome and phosphoproteome are specific to genotype and environment.•Loss of MLKs alters glucosinolate enzyme abundance and metabolism.•Loss of MLKs increases plant sensitivity to UV radiation and DNA damage agents.
The MUT9-like kinases are a family of plant-specific nuclear-localized kinases with roles in diverse signaling pathways, including light sensing, phytohormone perception, and the circadian clock. The proteome and phosphoproteome of compound mlk mutant seedlings have been determined under light and dark conditions. These experiments identify new roles for these kinases regulating secondary plant metabolism and stress responses, tested through metabolite analysis and assaying seedling sensitivity to DNA damaging agents.
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.
Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate ...reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution.
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.
•Terpene production did not affect plant morphology in the field.•Enhanced terpene lines performed better in the field than original lines.•Comparable lines produced more terpene in the greenhouse ...than field.•Terpene producing lines maintained stable oil composition under all conditions.•Cadinene lines had comparable seed yield to wild-type; limonene lines had less.
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.
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