ABSTRACT
Lonely guy (LOG) proteins are phosphoribohydrolases (PRHs) that are key cytokinin (CK)-activating enzymes in plant and non-plant CK-producing organisms. During CK biosynthesis, LOGs catalyze the conversion of precursor CK-nucleotides (CK-NTs) to biologically active free base forms. LOG/PRH activity has been detected in bacteria, archaea, algae, and fungi. However, in these organisms, the LOG/PRH activity for CK-NTs and non-CK-NTs (e.g., adenine-NTs) has not been assessed simultaneously, which leaves limited knowledge about the substrate specificity of LOGs. Thus, we performed bioinformatic analyses and a biochemical characterization of a LOG ortholog from Dictyostelium discoideum, a soil-dwelling amoeba, which produces CKs during unicellular growth and multicellular development. We show that DdLog exhibits LOG/PRH activity on two CK-NTs, N 6 -isopentenyladenosine-5'-monophosphate (iPMP) and N 6 -benzyladenosine-5'-monophosphate (BAMP), and on adenosine 5'-monophosphate (AMP) but not on 3', 5'-cyclic adenosine-monophosphate (cAMP). Additionally, there were higher turnover rates for CK-NTs over AMP. Together, these findings confirm that DdLog acts as a CK-activating enzyme; however, in contrast to plant LOGs, it maintains a wider specificity for other substrates (e.g., AMP) reflecting it has maintained its original, non-CK related role even after diversifying into a CK-activating enzyme.
ABSTRACT
Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.
Subject(s)
Euglena , Euglena/physiology , Biotechnology , SymbiosisABSTRACT
Euglena gracilis is a source of high-value natural products. A major factor affecting consistent production of Euglena biomass is strain stability. Cryopreservation is a leading strategy for cell-line storage that helps ensure process reproducibility. We developed a simple cryopreservation protocol for heterotrophically cultured Euglena that enables the recovery of cells after 1 year with a cell viability of â 80%. This protocol is suitable for labs interested in the long-term preservation of heterotrophic cultures of Euglena and related species.
Subject(s)
Euglena gracilis , Euglena , Biomass , Cryopreservation , Reproducibility of ResultsABSTRACT
To meet the growing demands of the oleochemical industry, tailored lipid sources are expanding to oleaginous microbes. To control the fatty acid composition of microbial lipids, ground-breaking exogenous and endogenous approaches are being developed. Exogenous approaches employ extracellular tools such as product-specific feedstocks, process optimization, elicitors, and magnetic and mechanical energy, whereas endogenous approaches leverage biology through the use of product-specific microbes, adaptive laboratory evolution (ALE), and the creation of custom strains via random and targeted cellular engineering. We consolidate recent advances from both fields into a review that will serve as a resource for those striving to fulfill the vision of microbial cell factories for tailored lipid production.
Subject(s)
Biofuels , Fatty AcidsABSTRACT
Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.
Subject(s)
Alkaloids/biosynthesis , Aspidosperma/chemistry , Tabernaemontana/chemistry , Alkaloids/chemistry , Carbazoles/chemistry , Cycloaddition Reaction/methods , Indole Alkaloids/chemistry , Plants/chemistryABSTRACT
(-)-Ibogaine and (-)-voacangine are plant derived psychoactives that show promise as treatments for opioid addiction. However, these compounds are produced by hard to source plants, making these chemicals difficult for broad-scale use. Here we report the complete biosynthesis of (-)-voacangine, and de-esterified voacangine, which is converted to (-)-ibogaine by heating, enabling biocatalytic production of these compounds. Notably, (-)-ibogaine and (-)-voacangine are of the opposite enantiomeric configuration compared to the other major alkaloids found in this natural product class. Therefore, this discovery provides insight into enantioselective enzymatic formal Diels-Alder reactions.
Subject(s)
Ibogaine/analogs & derivatives , Ibogaine/metabolism , Psychotropic Drugs/metabolism , Tabernaemontana/metabolism , Biosynthetic Pathways , Humans , Ibogaine/analysis , Opioid-Related Disorders/drug therapy , Psychotropic Drugs/analysis , Stereoisomerism , Tabernaemontana/chemistry , Tabernaemontana/enzymologyABSTRACT
Monoterpenoid indole alkaloids are a large (â¼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC-MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Ibogaine/biosynthesis , Protein O-Methyltransferase/metabolism , Tabernaemontana/chemistry , Alkaloids , Catalysis , High-Throughput Nucleotide Sequencing , Opioid-Related Disorders/drug therapy , Tabernaemontana/enzymology , Tabernaemontana/metabolism , Transcriptome/geneticsABSTRACT
Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.
Subject(s)
Antineoplastic Agents, Phytogenic/biosynthesis , Catharanthus/enzymology , Genes, Plant , Hydrolases/genetics , Vinblastine/biosynthesis , Antineoplastic Agents, Phytogenic/chemistry , Catharanthus/genetics , Indole Alkaloids/chemistry , Indole Alkaloids/metabolism , Quinolines/chemistry , Quinolines/metabolism , Vinblastine/chemistry , Vinca Alkaloids/biosynthesis , Vinca Alkaloids/chemistryABSTRACT
BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.
Subject(s)
Alkaloids/metabolism , Benzylisoquinolines/metabolism , Magnoliopsida/genetics , Plant Proteins/genetics , Transcriptome , Berberidaceae/genetics , Berberidaceae/metabolism , High-Throughput Nucleotide Sequencing , Magnoliopsida/metabolism , Menispermaceae/genetics , Menispermaceae/metabolism , Molecular Sequence Data , Papaveraceae/genetics , Papaveraceae/metabolism , Plant Proteins/metabolism , Ranunculaceae/genetics , Ranunculaceae/metabolism , Sequence Analysis, DNAABSTRACT
Opium poppy (Papaver somniferum) produces several pharmacologically important benzylisoquinoline alkaloids including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic origin of pacodine and palaudine has not been established. Three members of the 2-oxoglutarate/Fe(2+)-dependent dioxygenases (2ODDs) family in opium poppy display widespread O-dealkylation activity on several benzylisoquinoline alkaloids with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. We report a novel 2ODD from opium poppy catalyzing the efficient substrate- and regio-specific 7-O-demethylation of papaverine yielding pacodine. The occurrence of papaverine 7-O-demethylase (P7ODM) expands the enzymatic scope of the 2ODD family in opium poppy and suggests an unexpected biosynthetic route to pacodine.
Subject(s)
Iron/metabolism , Ketoglutaric Acids/metabolism , Oxidoreductases, O-Demethylating/metabolism , Papaver/enzymology , Papaverine/metabolism , Gene Expression Regulation, Plant , Methylation , Oxidoreductases, O-Demethylating/genetics , Oxidoreductases, O-Demethylating/isolation & purification , Papaverine/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stereoisomerism , Substrate SpecificityABSTRACT
The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.
Subject(s)
Aldehyde Reductase/metabolism , Carbohydrate Epimerases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Plant , Morphine/biosynthesis , Papaver/metabolism , Plant Proteins/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Alkaloids/biosynthesis , Alkaloids/chemistry , Base Sequence , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Bromoviridae/genetics , Bromoviridae/metabolism , Carbohydrate Epimerases/antagonists & inhibitors , Carbohydrate Epimerases/genetics , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Exons , Gene Fusion , Introns , Ligases/genetics , Ligases/metabolism , Molecular Sequence Data , Morphinans/chemistry , Morphinans/metabolism , Morphine/chemistry , Open Reading Frames , Opium/chemistry , Opium/metabolism , Oxidation-Reduction , Papaver/genetics , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , StereoisomerismABSTRACT
Oxidative enzymes catalyze many different reactions in plant metabolism. Among this suite of enzymes are the 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs). Cytochromes P450 (CYPs) as often considered the most versatile oxidative enzymes in nature, but the diversity and complexity of reactions catalyzed by 2-ODDs is superior to the CYPs. The list of oxidative reactions catalyzed by 2-ODDs includes hydroxylations, demethylations, desaturations, ring closure, ring cleavage, epimerization, rearrangement, halogenation, and demethylenation. Furthermore, recent work, including the discovery of 2-ODDs involved in epigenetic regulation, and others catalyzing several characteristic steps in specialized metabolic pathways, support the argument that 2-ODDs are among the most versatile and important oxidizing biological catalysts. In this review, we survey and summarize the pertinent literature with a focus on several key reactions catalyzed by 2-ODDs, and discuss the significance and impact of these enzymes in plant metabolism.
ABSTRACT
In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.
Subject(s)
Benzylisoquinolines/metabolism , Biocatalysis , Dioxygenases/metabolism , Opium/metabolism , Papaver/enzymology , Benzylisoquinolines/chemistry , Berberine Alkaloids/chemistry , Berberine Alkaloids/metabolism , Chromatography, Liquid , Formaldehyde/metabolism , Gene Silencing , Kinetics , Mass Spectrometry , Methylation , Phylogeny , Substrate Specificity , VirusesABSTRACT
BACKGROUND: Cytokinins (CKs) are a group of plant growth regulators that are involved in several plant developmental processes. Despite the breadth of knowledge surrounding CKs and their diverse functions, much remains to be discovered about the full potential of CKs, including their relationship with the purine salvage pathway, and other phytohormones. The most widely used approach to query unknown facets of CK biology utilized functional genomics coupled with CK metabolite assays and screening of CK associated phenotypes. There are numerous different types of assays for determining CK quantity, however, none of these methods screen for the compendium of metabolites that are necessary for elucidating all roles, including purine salvage pathway enzymes in CK metabolism, and CK cross-talk with other phytohormones. Furthermore, all published analytical methods have drawbacks ranging from the required use of radiolabelled compounds, or hazardous derivatization reagents, poor sensitivity, lack of resolution between CK isomers and lengthy run times. RESULTS: In this paper, a method is described for the concurrent extraction, purification and analysis of several CKs (freebases, ribosides, glucosides, nucleotides), purines (adenosine monophosphate, inosine, adenosine, and adenine), indole-3-acetic acid, and abscisic acid from hundred-milligram (mg) quantities of Arabidopsis thaliana leaf tissue. This method utilizes conventional Bieleski solvents extraction, solid phase purification, and is unique because of its diverse range of detectable analytes, and implementation of a conventional HPLC system with a fused core column that enables good sensitivity without the requirement of a UHPLC system. Using this method we were able to resolve CKs about twice as fast as our previous method. Similarly, analysis of adenosine, indole-3-acetic acid, and abscisic acid, was comparatively rapid. A further enhancement of the method was the utilization of a QTRAP 5500 mass analyzer, which improved upon several aspects of our previous analytical method carried out on a Quattro mass analyzer. Notable improvements included much superior sensitivity, and number of analytes detectable within a single run. Limits of detection ranged from 2 pM for (9G)Z to almost 750 pM for indole-3-acetic acid. CONCLUSIONS: This method is well suited for functional genomics platforms tailored to understanding CK metabolism, CK interrelationships with purine recycling and associated hormonal cross-talk.
ABSTRACT
Benzylisoquinoline alkaloids (BIAs) are a large, diverse group of â¼2500 specialized plant metabolites. Many BIAs display potent pharmacological activities, including the narcotic analgesics codeine and morphine, the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine, the antimicrobial agents sanguinarine and berberine, and the muscle relaxant (+)-tubocurarine. Opium poppy remains the sole commercial source for codeine, morphine, and a variety of semisynthetic drugs, including oxycodone and buprenorphine, derived primarily from the biosynthetic pathway intermediate thebaine. Recent advances in transcriptomics, proteomics, and metabolomics have created unprecedented opportunities for isolating and characterizing novel BIA biosynthetic genes. Here, we describe the application of next-generation sequencing and cDNA microarrays for selecting gene candidates based on comparative transcriptome analysis. We outline the basic mass spectrometric techniques to perform deep proteome and targeted metabolite analyses on BIA-producing plant tissues and provide methodologies for functionally characterizing biosynthetic gene candidates through in vitro enzyme assays and transient gene silencing in planta.
Subject(s)
Benzylisoquinolines/chemistry , Genomics/methods , Papaver/chemistry , Papaver/genetics , Biosynthetic Pathways , Electrophoresis, Polyacrylamide Gel , Enzyme Assays , Gene Expression Profiling , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant , Mass Spectrometry , Metabolomics/methods , Oligonucleotide Array Sequence Analysis , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/isolation & purification , Tandem Mass SpectrometryABSTRACT
Recent advances in DNA sequencing technology and analytical mass spectrometry are providing unprecedented opportunities to develop the functional genomics resources required to investigate complex biological processes in non-model plants. Opium poppy produces a wide variety of benzylisoquinoline alkaloids (BIAs), including the pharmaceutical compounds codeine, morphine, noscapine and papaverine. A functional genomics platform to identify novel BIA biosynthetic and regulatory genes in opium poppy has been established based on the differential metabolite profile of eight selected cultivars. Stem cDNA libraries from each of the eight opium poppy cultivars were subjected to 454 pyrosequencing and searchable expressed sequence tag databases were created from the assembled reads. These deep and integrated metabolite and transcript databases provide a nearly complete representation of the genetic and metabolic variances responsible for the differential occurrence of specific BIAs in each cultivar as demonstrated using the biochemically well characterized pathway from tyrosine to morphine. Similar correlations between the occurrence of specific transcripts and alkaloids effectively reveals candidate genes encoding uncharacterized biosynthetic enzymes as shown using cytochromes P450 potentially involved in the formation of papaverine and noscapine.
Subject(s)
Benzylisoquinolines/metabolism , Papaver/metabolism , Gene Expression Regulation, Plant , Papaver/geneticsABSTRACT
Benzylisoquinoline alkaloids (BIAs) are a large and diverse group of ~2500 specialized metabolites found predominantly in plants of the order Ranunculales. Research focused on BIA metabolism in a restricted number of plant species has identified many enzymes and cognate genes involved in the biosynthesis of compounds such as morphine, sanguinarine and berberine. However, the formation of most BIAs remains uncharacterized at the molecular biochemical level. Herein a compendium of sequence- and metabolite-profiling resources from 18 species of BIA-accumulating cell cultures was established, representing four related plant families. Our integrated approach consisted of the construction of EST libraries each containing approximately 3500 unigenes per species for a total of 58,787 unigenes. The EST libraries were manually triaged using known BIA-biosynthetic genes as queries to identify putative homologs with similar or potentially different functions. Sequence resources were analyzed in the context of the targeted metabolite profiles obtained for each cell culture using electrospray-ionization and collision-induced dissociation mass spectrometry. Fragmentation analysis was used for the identification or structural characterization coupled with the relative quantification of 72 BIAs, which establishes a key resource for future work on alkaloid biosynthesis. The metabolite profile obtained for each species provides a rational basis for the prediction of enzyme function in BIA metabolism. The metabolic frameworks assembled through the integration of transcript and metabolite profiles allow a comparison of BIA metabolism across several plant species and families. Taken together, these data represent an important tool for the discovery of BIA biosynthetic genes.
Subject(s)
Alkaloids/chemistry , Benzylisoquinolines/chemistry , Ranunculaceae/metabolism , Alkaloids/biosynthesis , Alkaloids/metabolism , Benzylisoquinolines/metabolism , Cells, Cultured , Databases, Nucleic Acid , Expressed Sequence Tags , RNA, Messenger/chemistry , Ranunculaceae/geneticsABSTRACT
Purine salvage enzymes have been implicated, but not proven, to be involved in the interconversion of cytokinin (CK) bases, ribosides, and nucleotides. Here, we use Arabidopsis (Arabidopsis thaliana) lines silenced in adenosine kinase (ADK) expression to understand the contributions of this enzyme activity to in vivo CK metabolism. Both small interfering RNA- and artificial microRNA-mediated silencing of ADK led to impaired root growth, small, crinkled rosette leaves, and reduced apical dominance. Further examination of ADK-deficient roots and leaves revealed their irregular cell division. Root tips had uneven arrangements of root cap cells, reduced meristem sizes, and enlarged cells in the elongation zone; rosette leaves exhibited decreased cell size but increased cell abundance. Expression patterns of the cyclinB1;1::ß-glucuronidase and Arabidopsis Response Regulator5::ß-glucuronidase reporters in the ADK-deficient background were consistent with altered cell division and an increase in CK activity, respectively. In vivo feeding of ADK-deficient leaves with radiolabeled CK ribosides of isopentenyladenosine and zeatin showed a decreased flux into the corresponding CK nucleotides. Comprehensive high-performance liquid chromatography-tandem mass spectrometry analysis detected significantly higher levels of active CK ribosides in both sense ADK and artificial microADK. Taken together, these metabolic and phenotypic analyses of ADK-deficient lines indicate that ADK contributes to CK homeostasis in vivo.
Subject(s)
Adenosine Kinase/metabolism , Arabidopsis/metabolism , Cytokinins/metabolism , Adenosine Kinase/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclin B1/genetics , Cyclin B1/metabolism , Cytokinins/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Glucuronidase/genetics , Glucuronidase/metabolism , Homozygote , Meristem/cytology , Meristem/growth & development , MicroRNAs , Plant Leaves/growth & development , Plant Roots/growth & development , RNA Interference , Transcription Factors/genetics , Zeatin/genetics , Zeatin/metabolismABSTRACT
The analysis of federal regulations focuses on the analysis of economic efficiency through standard benefit-cost analysis in which reductions in risk tend to represent benefits and variability may be investigated through sensitivity or simulation methods. Such analyses typically ignore distributional consequences that can affect decisions. Although analytical guidance from the Office of Management and Budget (OMB) mentions distributional consequences, there is little specificity regarding how it might be analyzed. Specificity might be improved by OMB: (1) communicating and enforcing distributional impacts as more central to the process, (2) identifying default types of descriptive distributional statistics, (3) identifying whether legislation has created a constraint to not harm a subgroup, and (4) requesting a distributional sensitivity test of the net benefits along with a standard benefit-cost analysis. Although such actions have a data collection and analysis cost, they may make the results of regulatory analysis more relevant by investigating both efficiency and equity measures.