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Birger Lindberg Moller - One of the best experts on this subject based on the ideXlab platform.
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the interplay between water limitation Dhurrin and nitrate in the low cyanogenic sorghum mutant adult cyanide deficient class 1
Frontiers in Plant Science, 2019Co-Authors: Viviana C Rosati, Birger Lindberg Moller, Cecilia K Blomstedt, Trevor Garnett, Roslyn M GleadowAbstract:Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product Dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase Dhurrin in shoot tissues to concentrations toxic to livestock. As Dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in Dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low Dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and Dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of Dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased Dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower Dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate Dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.
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Crop wild relatives as a genetic resource for generating low-cyanide, drought-tolerant Sorghum
Environmental and Experimental Botany, 2019Co-Authors: Max F. Cowan, Birger Lindberg Moller, Cecilia K Blomstedt, Sally L. Norton, Robert J Henry, Roslyn M GleadowAbstract:Abstract Using a Sorghum bicolor cultivar and seven wild Sorghum species endemic to Australia as our experimental system, we monitored their different responses to drought by assessing growth and morphological, physiological and biochemical parameters. Drought stress significantly decreased height, biomass, the maximum potential quantum efficiency of photosystem II, photosynthetic rate and relative water content in S. bicolor, while several of the wild species were much more tolerant. Drought significantly increased Dhurrin concentration in aboveground tissue in S. bicolor but not in the wild species. Root Dhurrin content was unaffected by drought in S. bicolor, in contrast to the varied responses observed in the wild species. Sorghum macrospermum and S. brachypodum maintained relatively high growth and photosynthetic performance under drought, with negligible aboveground Dhurrin content. These wild species are promising candidates for sorghum crop improvement.
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Counting the costs: nitrogen partitioning in Sorghum mutants.
Functional Plant Biology, 2018Co-Authors: Cecilia K Blomstedt, Birger Lindberg Moller, Viviana C Rosati, Roslyn M GleadowAbstract:Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside Dhurrin, with unique mutants that vary in Dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise Dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and Dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that Dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and Dhurrin suggests these may be independently regulated.
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label free raman hyperspectral imaging analysis localizes the cyanogenic glucoside Dhurrin to the cytoplasm in sorghum cells
Scientific Reports, 2018Co-Authors: Philip Heraud, Birger Lindberg Moller, Cecilia K Blomstedt, Max F. Cowan, Katarzyna M Marzec, Roslyn M GleadowAbstract:Localisation of metabolites in sorghum coleoptiles using Raman hyperspectral imaging analysis was compared in wild type plants and mutants that lack cyanogenic glucosides. This novel method allows high spatial resolution in situ localization by detecting functional groups associated with cyanogenic glucosides using vibrational spectroscopy. Raman hyperspectral imaging revealed that Dhurrin was found mainly surrounding epidermal, cortical and vascular tissue, with the greatest amount in cortical tissue. Numerous “hotspots” demonstrated Dhurrin to be located within both cell walls and cytoplasm adpressed towards the plasmamembrane and not in the vacuole as previously reported. The high concentration of Dhurrin in the outer cortical and epidermal cell layers is consistent with its role in defence against herbivory. This demonstrates the ability of Raman hyperspectral imaging to locate cyanogenic glucosides in intact tissues, avoiding possible perturbations and imprecision that may accompany methods that rely on bulk tissue extraction methods, such as protoplast isolation.
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Assembly of Dynamic P450-Mediated Metabolons—Order Versus Chaos
Current Molecular Biology Reports, 2017Co-Authors: Jean-etienne Bassard, Birger Lindberg Moller, Tomas LaursenAbstract:Purpose of Review We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the Dhurrin metabolon from Sorghum bicolor as a case study. Recent Findings Sessile plants control their metabolism to prioritize their resources between growth and development, or defense. This requires fine-tuned complex dynamic regulation of the metabolic networks involved. Within the recent years, numerous studies point to the formation of dynamic metabolons playing a major role in controlling the metabolic fluxes within such networks. Summary We propose that P450s and their partners interact and associate dynamically with POR, which acts as a charging station possibly in concert with Cyt b5 . Solvent environment, lipid composition, and non-catalytic proteins guide metabolon formation and thereby activity, which have important implications for synthetic biology approaches aiming to produce high-value specialized metabolites in heterologous hosts.
Barbara Ann Halkier - One of the best experts on this subject based on the ideXlab platform.
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transgenic tobacco and arabidopsis plants expressing the two multifunctional sorghum cytochrome p450 enzymes cyp79a1 and cyp71e1 are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesis
Plant Physiology, 2000Co-Authors: Carl Erik Olsen, Barbara Ann Halkier, Birger Lindberg MollerAbstract:Novel cyanogenic plants have been generated by the simultaneous expression of the two multifunctional sorghum (Sorghum bicolor [L.] Moench) cytochrome P450 enzymes CYP79A1 and CYP71E1 in tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis under the regulation of the constitutive 35S promoter. CYP79A1 and CYP71E1 catalyze the conversion of the parent amino acid tyrosine to p-hydroxymandelonitrile, the aglycone of the cyanogenic glucoside Dhurrin. CYP79A1 catalyzes the conversion of tyrosine to p-hydroxyphenylacetaldoxime and CYP71E1, the subsequent conversion to p-hydroxymandelonitrile. p-Hydroxymandelonitrile is labile and dissociates into p-hydroxybenzaldehyde and hydrogen cyanide, the same products released from Dhurrin upon cell disruption as a result of pest or herbivore attack. In transgenic plants expressing CYP79A1 as well as CYP71E1, the activity of CYP79A1 is higher than that of CYP71E1, resulting in the accumulation of several p-hydroxyphenylacetaldoxime-derived products in the addition to those derived from p-hydroxymandelonitrile. Transgenic tobacco and Arabidopsis plants expressing only CYP79A1 accumulate the same p-hydroxyphenylacetaldoxime-derived products as transgenic plants expressing both sorghum cytochrome P450 enzymes. In addition, the transgenic CYP79A1 Arabidopsis plants accumulate large amounts of p-hydroxybenzyl glucosinolate. In transgenic Arabidopsis expressing CYP71E1, this enzyme and the enzymes of the pre-existing glucosinolate pathway compete for the p-hydroxyphenylacetaldoxime as substrate, resulting in the formation of small amounts of p-hydroxybenzylglucosinolate. Cyanogenic glucosides are phytoanticipins, and the present study demonstrates the feasibility of expressing cyanogenic compounds in new plant species by gene transfer technology to improve pest and disease resistance.
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cloning of three a type cytochromes p450 cyp71e1 cyp98 and cyp99 from sorghum bicolor l moench by a pcr approach and identification by expression in escherichia coli of cyp71e1 as a multifunctional cytochrome p450 in the biosynthesis of the cyanogeni
Plant Molecular Biology, 1998Co-Authors: Rachel Alice Kahn, Birger Lindberg Moller, Hanne Linde Nielsen, Barbara Ann HalkierAbstract:A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside Dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.
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cloning of three a type cytochromes p450 cyp71e1 cyp98 and cyp99 from sorghum bicolor l moench by a pcr approach and identification by expression in escherichia coli of cyp71e1 as a multifunctional cytochrome p450 in the biosynthesis of the cyanogeni
Plant Molecular Biology, 1998Co-Authors: Rachel Alice Kahn, Birger Lindberg Moller, Hanne Linde Nielsen, Barbara Ann HalkierAbstract:A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside Dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L-α-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in Dhurrin biosynthesis. In accordance to the proposed pathway for Dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside Dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside Dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.
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Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside Dhurrin from sorghum.
Plant physiology, 1997Co-Authors: Rache Alice Kahn, Slsren Bak, Barbara Ann Halkier, Ib Svendsen, B L MøllerAbstract:A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (Dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to Dhurrin. In vitro reconstitution of the entire Dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.
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the biosynthesis of cyanogenic glucosides in higher plants identification of three hydroxylation steps in the biosynthesis of Dhurrin in sorghum bicolor l moench and the involvement of 1 aci nitro 2 p hydroxyphenyl ethane as an intermediate
Journal of Biological Chemistry, 1990Co-Authors: Barbara Ann Halkier, B L MøllerAbstract:N-Hydroxytyrosine, (E)- and (Z)-p-hydroxyphenyl-acetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile are established intermediates in the biosynthesis of the tyrosine-derived cyanogenic glucoside Dhurrin. Simultaneous measurements of oxygen consumption and biosynthetic activity using a microsomal enzyme system isolated from etiolated sorghum seedlings demonstrate a requirement for three oxygen molecules in the conversion of tyrosine to p-hydroxymandelonitrile. Two oxygen molecules are consumed in the conversion of tyrosine to (E)-p-hydroxyphenylacetaldehyde oxime, indicating the existence of a previously undetected hydroxylation step in addition to that resulting in the formation of N-hydroxytyrosine. Radioactively labeled 1-nitro-2-(p-hydroxyphenyl)ethane was chemically synthesized and tested as a possible intermediate. Biosynthetic experiments demonstrate that the microsomal enzyme system metabolizes the nitro compound to the subsequent intermediates in Dhurrin synthesis (Km = 0.05 mM; Vmax = 14 nmol/mg of protein/h). Low amounts of 1-nitro-2-(p-hydroxyphenyl)ethane are produced in the microsomal reaction mixtures when tyrosine is used as substrate. These data support the involvement of 1-nitro-2-(p-hydroxyphenyl)ethane or more likely its aci-nitro tautomer as an intermediate between N-hydroxytyrosine and p-hydroxyphenylacetaldehyde oxime. The conversion of (E)-p-hydroxyphenylacetaldehydeoxime to p-hydroxymandelonitrile requires a single oxygen molecule. The oxygen molecule is utilized for hydroxylation of p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. This indicates that the conversion of p-hydroxyphenylacetaldehyde oxime into p-hydroxyphenylacetonitrile proceeds by a simple dehydrationmore » reaction.« less
Carl Erik Olsen - One of the best experts on this subject based on the ideXlab platform.
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Dhurrin metabolism in the developing grain of Sorghum bicolor (L.) Moench investigated by metabolite profiling and novel clustering analyses of time-resolved transcriptomic data
BMC Genomics, 2016Co-Authors: Lasse Janniche Nielsen, Birger Lindberg Moller, Carl Erik Olsen, Peter Stuart, Martina Pičmanová, Simon Rasmussen, Jesper Harholt, Nanna BjarnholtAbstract:Background The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound Dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of Dhurrin for which putative pathways have been suggested but not confirmed. Results In this study, the biosynthesis and endogenous turnover of Dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the Dhurrin content was turned over, resulting in only negligible residual Dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of Dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding Dhurrinases. With the missing activity of Dhurrinases, the decrease in Dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous Dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of Dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum. Conclusions The results presented in this article reveal the existence of two endogenous Dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that Dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace Dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.
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Dhurrin metabolism in the developing grain of sorghum bicolor l moench investigated by metabolite profiling and novel clustering analyses of time resolved transcriptomic data
BMC Genomics, 2016Co-Authors: Lasse Janniche Nielsen, Birger Lindberg Moller, Carl Erik Olsen, Peter Stuart, Martina Pičmanová, Simon Rasmussen, Jesper Harholt, Nanna BjarnholtAbstract:The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound Dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of Dhurrin for which putative pathways have been suggested but not confirmed. In this study, the biosynthesis and endogenous turnover of Dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the Dhurrin content was turned over, resulting in only negligible residual Dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of Dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding Dhurrinases. With the missing activity of Dhurrinases, the decrease in Dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous Dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of Dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum. The results presented in this article reveal the existence of two endogenous Dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that Dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace Dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.
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the biosynthetic gene cluster for the cyanogenic glucoside Dhurrin in sorghum bicolor contains its co expressed vacuolar mate transporter
Scientific Reports, 2016Co-Authors: Behrooz Darbani, Birger Lindberg Moller, Carl Erik Olsen, Mohammed Saddik Motawia, Hussam Hassan Noureldin, Fred RookAbstract:Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside Dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport Dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.
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transfer of the cytochrome p450 dependent Dhurrin pathway from sorghum bicolor into nicotiana tabacum chloroplasts for light driven synthesis
Journal of Experimental Botany, 2016Co-Authors: Thiyagarajan Gnanasekaran, Agnieszka Zygadlo Nielsen, Helle Juel Martens, Xenia Kroop, Mohammed Saddik Motawie, Daniel Karcher, Mathias Pribil, Birger Lindberg Moller, Carl Erik Olsen, Ralph BockAbstract:Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the Dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into Dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The Dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.
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Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.
Metabolic Engineering, 2015Co-Authors: Artur Włodarczyk, Thiyagarajan Gnanasekaran, Agnieszka Zygadlo Nielsen, Carl Erik Olsen, Nodumo Nokolunga Zulu, Silas Busck Mellor, Manja Luckner, Jens Frederik Bang Thøfner, Mohammed Saddik Mottawie, Meike BurowAbstract:Abstract Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the Dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo . The engineered strains produced up to 66 mg/L of p -hydroxyphenylacetaldoxime and 5 mg/L of Dhurrin in lab-scale cultures after 3 days of cultivation and 3 mg/L of Dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.
Roslyn M Gleadow - One of the best experts on this subject based on the ideXlab platform.
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Matrix-assisted laser desorption/ionization-mass spectrometry imaging of metabolites during sorghum germination.
Plant Physiology, 2020Co-Authors: Lucia Montini, Roslyn M Gleadow, Christoph Crocoll, Mohammed Saddik Motawia, Christian Janfelt, Nanna BjarnholtAbstract:Dhurrin is the most abundant cyanogenic glucoside found in sorghum (Sorghum bicolor) where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that Dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why Dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of Dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no Dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of Dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that Dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The Dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination.
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the interplay between water limitation Dhurrin and nitrate in the low cyanogenic sorghum mutant adult cyanide deficient class 1
Frontiers in Plant Science, 2019Co-Authors: Viviana C Rosati, Birger Lindberg Moller, Cecilia K Blomstedt, Trevor Garnett, Roslyn M GleadowAbstract:Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product Dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase Dhurrin in shoot tissues to concentrations toxic to livestock. As Dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in Dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low Dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and Dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of Dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased Dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower Dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate Dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.
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Crop wild relatives as a genetic resource for generating low-cyanide, drought-tolerant Sorghum
Environmental and Experimental Botany, 2019Co-Authors: Max F. Cowan, Birger Lindberg Moller, Cecilia K Blomstedt, Sally L. Norton, Robert J Henry, Roslyn M GleadowAbstract:Abstract Using a Sorghum bicolor cultivar and seven wild Sorghum species endemic to Australia as our experimental system, we monitored their different responses to drought by assessing growth and morphological, physiological and biochemical parameters. Drought stress significantly decreased height, biomass, the maximum potential quantum efficiency of photosystem II, photosynthetic rate and relative water content in S. bicolor, while several of the wild species were much more tolerant. Drought significantly increased Dhurrin concentration in aboveground tissue in S. bicolor but not in the wild species. Root Dhurrin content was unaffected by drought in S. bicolor, in contrast to the varied responses observed in the wild species. Sorghum macrospermum and S. brachypodum maintained relatively high growth and photosynthetic performance under drought, with negligible aboveground Dhurrin content. These wild species are promising candidates for sorghum crop improvement.
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investigation into the role of dna methylation in cyanogenesis in sorghum sorghum bicolor l moench
Plant Growth Regulation, 2019Co-Authors: Viviana C Rosati, Roslyn M Gleadow, Alicia A Quinn, Samantha M Fromhold, Cecilia K BlomstedtAbstract:Sorghum bicolor produces the cyanogenic glucoside Dhurrin, a secondary metabolite integral to plant defence and stress responses. Dhurrin production is both developmentally and environmentally regulated in S. bicolor, with high levels of variation within and between lines. Such phenotypic variation may result from polymorphic differences or epigenetic modifications in genes associated with cyanogenesis. In this study the chemical 5-Azacytidine was used to assess S. bicolor’s response to genome-wide demethylation, which had not previously been investigated in the context of cyanogenic glucoside regulation. Morphological changes, the expression levels of key genes involved in Dhurrin synthesis and turnover, and the cyanogenic potential (HCNp) of leaf tissues were analysed. Treatment resulted in alterations in Dhurrin synthesis, gene expression, and Dhurrin levels, suggesting that DNA methylation is involved in the regulation of HCNp in the initial stages of S. bicolor development. Previously identified EMS mutants from the adult cyanide deficient class (acdc) have been found to exhibit altered Dhurrin concentrations during development. This study shows that acdc mutants possess a CΔT change in the promoter of CYP79A1, a key gene in Dhurrin synthesis, and that this mutation is stably inherited and associated with the acdc phenotype. To further investigate the role of epigenesis in Dhurrin production, we determine the methylation status of the 250 bp region surrounding the CΔT mutation site in wild-type and mutant plants over two stages of development.
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Counting the costs: nitrogen partitioning in Sorghum mutants.
Functional Plant Biology, 2018Co-Authors: Cecilia K Blomstedt, Birger Lindberg Moller, Viviana C Rosati, Roslyn M GleadowAbstract:Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside Dhurrin, with unique mutants that vary in Dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise Dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and Dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that Dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and Dhurrin suggests these may be independently regulated.
Cecilia K Blomstedt - One of the best experts on this subject based on the ideXlab platform.
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the interplay between water limitation Dhurrin and nitrate in the low cyanogenic sorghum mutant adult cyanide deficient class 1
Frontiers in Plant Science, 2019Co-Authors: Viviana C Rosati, Birger Lindberg Moller, Cecilia K Blomstedt, Trevor Garnett, Roslyn M GleadowAbstract:Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product Dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase Dhurrin in shoot tissues to concentrations toxic to livestock. As Dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in Dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low Dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and Dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of Dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased Dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower Dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate Dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.
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Crop wild relatives as a genetic resource for generating low-cyanide, drought-tolerant Sorghum
Environmental and Experimental Botany, 2019Co-Authors: Max F. Cowan, Birger Lindberg Moller, Cecilia K Blomstedt, Sally L. Norton, Robert J Henry, Roslyn M GleadowAbstract:Abstract Using a Sorghum bicolor cultivar and seven wild Sorghum species endemic to Australia as our experimental system, we monitored their different responses to drought by assessing growth and morphological, physiological and biochemical parameters. Drought stress significantly decreased height, biomass, the maximum potential quantum efficiency of photosystem II, photosynthetic rate and relative water content in S. bicolor, while several of the wild species were much more tolerant. Drought significantly increased Dhurrin concentration in aboveground tissue in S. bicolor but not in the wild species. Root Dhurrin content was unaffected by drought in S. bicolor, in contrast to the varied responses observed in the wild species. Sorghum macrospermum and S. brachypodum maintained relatively high growth and photosynthetic performance under drought, with negligible aboveground Dhurrin content. These wild species are promising candidates for sorghum crop improvement.
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investigation into the role of dna methylation in cyanogenesis in sorghum sorghum bicolor l moench
Plant Growth Regulation, 2019Co-Authors: Viviana C Rosati, Roslyn M Gleadow, Alicia A Quinn, Samantha M Fromhold, Cecilia K BlomstedtAbstract:Sorghum bicolor produces the cyanogenic glucoside Dhurrin, a secondary metabolite integral to plant defence and stress responses. Dhurrin production is both developmentally and environmentally regulated in S. bicolor, with high levels of variation within and between lines. Such phenotypic variation may result from polymorphic differences or epigenetic modifications in genes associated with cyanogenesis. In this study the chemical 5-Azacytidine was used to assess S. bicolor’s response to genome-wide demethylation, which had not previously been investigated in the context of cyanogenic glucoside regulation. Morphological changes, the expression levels of key genes involved in Dhurrin synthesis and turnover, and the cyanogenic potential (HCNp) of leaf tissues were analysed. Treatment resulted in alterations in Dhurrin synthesis, gene expression, and Dhurrin levels, suggesting that DNA methylation is involved in the regulation of HCNp in the initial stages of S. bicolor development. Previously identified EMS mutants from the adult cyanide deficient class (acdc) have been found to exhibit altered Dhurrin concentrations during development. This study shows that acdc mutants possess a CΔT change in the promoter of CYP79A1, a key gene in Dhurrin synthesis, and that this mutation is stably inherited and associated with the acdc phenotype. To further investigate the role of epigenesis in Dhurrin production, we determine the methylation status of the 250 bp region surrounding the CΔT mutation site in wild-type and mutant plants over two stages of development.
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Counting the costs: nitrogen partitioning in Sorghum mutants.
Functional Plant Biology, 2018Co-Authors: Cecilia K Blomstedt, Birger Lindberg Moller, Viviana C Rosati, Roslyn M GleadowAbstract:Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside Dhurrin, with unique mutants that vary in Dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise Dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and Dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that Dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and Dhurrin suggests these may be independently regulated.
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label free raman hyperspectral imaging analysis localizes the cyanogenic glucoside Dhurrin to the cytoplasm in sorghum cells
Scientific Reports, 2018Co-Authors: Philip Heraud, Birger Lindberg Moller, Cecilia K Blomstedt, Max F. Cowan, Katarzyna M Marzec, Roslyn M GleadowAbstract:Localisation of metabolites in sorghum coleoptiles using Raman hyperspectral imaging analysis was compared in wild type plants and mutants that lack cyanogenic glucosides. This novel method allows high spatial resolution in situ localization by detecting functional groups associated with cyanogenic glucosides using vibrational spectroscopy. Raman hyperspectral imaging revealed that Dhurrin was found mainly surrounding epidermal, cortical and vascular tissue, with the greatest amount in cortical tissue. Numerous “hotspots” demonstrated Dhurrin to be located within both cell walls and cytoplasm adpressed towards the plasmamembrane and not in the vacuole as previously reported. The high concentration of Dhurrin in the outer cortical and epidermal cell layers is consistent with its role in defence against herbivory. This demonstrates the ability of Raman hyperspectral imaging to locate cyanogenic glucosides in intact tissues, avoiding possible perturbations and imprecision that may accompany methods that rely on bulk tissue extraction methods, such as protoplast isolation.