RESUMEN
Plant breeders leverage mutagenesis using chemical, biological, and physical mutagens to create novel trait variations. Many widely used sorghum genotypes have a narrow genetic base, which hinders improvements using classical breeding. Enhancing the diversity of the sorghum genome thus remains a key priority for sorghum breeders. To accelerate the genetic enhancement of sorghum, an extensive library comprised of seeds from 150,000 individual mutant plants of the Sorghum bicolor inbred line BTx623 was established using ethyl methanesulphonate (EMS) as a mutagen. The sorghum mutant library was bulked into 1498 pools (~100 seed heads per pool). In each pool, DNA was extracted from a subset of the seed and screened using the FIND-IT technology based on droplet digital PCR. All 43 nucleotide substitutions that were screened using FIND-IT were identified, demonstrating the potential to identify any EMS-derived mutation in an elite line of sorghum within days. This diverse library represents the largest collection of sorghum mutants ever conceived, estimated to cover 240% of all possible EMS-induced mutation points within the Sorghum genome. Using FIND-IT, the speed at which a specific desired EMS-derived mutation can be identified is a major upgrade to conventional reverse genetic techniques. Additionally, the ease at which valuable variants can be integrated into elite commercial lines is a far simpler and less expensive process compared to genome editing. Genomic variations in the library will have direct utility as a breeding resource for commercial sorghum applications, allowing enhanced adaptation to climate change and enhanced yield potential in marginal environments.
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Metanosulfonato de Etilo , Mutagénesis , Fitomejoramiento , Sorghum , Sorghum/genética , Sorghum/efectos de los fármacos , Mutagénesis/genética , Fitomejoramiento/métodos , Mutación/genética , Genotipo , Productos Agrícolas/genética , Genoma de Planta/genética , Semillas/genética , Semillas/efectos de los fármacos , Mutágenos , Biblioteca de GenesRESUMEN
INTRODUCTION: In recent years, industrial production of Cannabis sativa has increased due to increased demand of medicinal products based on the plant. In these medicinal products, it is mainly the contents of cannabinoids like THCA and CBDA which are of interest, but also the flavonoids of C. sativa have pharmaceutical interest. OBJECTIVES: The primary aim is to study the distribution of the different cannabinoids in leaves of C. sativa and specifically to which extent they are located on the trichomes found on the surface of C. sativa leaves. Desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) provide non-targeted imaging of numerous compounds in the same experiment. Therefore, the distribution of flavonoids is also mapped in the same experiments. MATERIAL AND METHODS: Fan leaves from C. sativa were imaged in the lateral dimension using direct DESI-MSI as well as indirect DESI-MSI via a porous PTFE surface using pixel sizes of 150-200 µm. For cross sections of sugar leaves, MALDI-MSI was performed at 20 µm pixel size. RESULTS: From indirect DESI-MSI experiments, a connection was made between the cannabinoid CBGA and capitate-stalked trichomes. Other cannabinoids like THCA/CBDA (isomers, which are not resolved in an MSI experiment) were also detected in the capitate-stalked trichomes, but in addition to this also in the small glandular trichomes. MALDI-MSI experiments on cross sections of sugar leaves confirmed that the cannabinoids were not an integral part of the leaf tissue itself, but originated from the trichomes on the surface of the leaf. CONCLUSION: The study provides visual evidence that the cannabinoids are produced and accumulated in the trichomes of C. sativa leaves.
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Cannabinoides , Cannabis , Cannabinoides/análisis , Cannabis/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tricomas/química , Flavonoides/análisis , Hojas de la Planta/química , Azúcares/análisisRESUMEN
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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Germinación/fisiología , Nitrilos/metabolismo , Semillas/genética , Semillas/fisiología , Sorghum/genética , Sorghum/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Grano Comestible/genética , Grano Comestible/fisiología , Regulación de la Expresión Génica de las Plantas , Germinación/genéticaRESUMEN
Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.
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Pared Celular/química , Fenoles/química , Plantas/química , Secuencia de CarbohidratosRESUMEN
Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.
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Glutatión Transferasa/metabolismo , Glicósidos/metabolismo , Proteínas de Plantas/metabolismo , Sorghum/enzimología , Catálisis , Cianuro de Hidrógeno/metabolismo , Redes y Vías Metabólicas , Nitrilos/metabolismo , Sorghum/metabolismoRESUMEN
The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/ß glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish α- and ß-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.
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Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the ß-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized ß-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.
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Arabidopsis/genética , Arabidopsis/metabolismo , Lignina/metabolismo , Sphingomonas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica de las Plantas , Ingeniería Genética/métodos , Glucosa/metabolismo , Lignina/química , Espectroscopía de Resonancia Magnética , Redes y Vías Metabólicas/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
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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Metaboloma , Metabolómica , Nitrilos/metabolismo , Sorghum/genética , Sorghum/metabolismo , Transcriptoma , Análisis por Conglomerados , Cianuros/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glutatión Transferasa/metabolismo , Metabolómica/métodos , Filogenia , Proantocianidinas/metabolismo , Semillas/genética , Semillas/metabolismo , Sorghum/clasificación , Sorghum/crecimiento & desarrolloRESUMEN
Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.
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Técnicas de Inactivación de Genes , Genes de Plantas , Glucosiltransferasas/genética , Nitrilos/metabolismo , Sorghum/genética , Sorghum/metabolismo , Cromatografía Liquida , Glucosiltransferasas/metabolismo , Cianuro de Hidrógeno/metabolismo , Espectrometría de Masas , Metaboloma , Metabolómica , Mutación/genética , Nitratos/metabolismo , Nitrilos/química , Nitrógeno/metabolismo , Fenotipo , Plantas Modificadas Genéticamente , Sorghum/enzimología , Sorghum/crecimiento & desarrolloRESUMEN
Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and high resolution MS (HR-MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and 'anitriles' derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.
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Glicósidos/metabolismo , Manihot/metabolismo , Prunus/metabolismo , Sorghum/metabolismo , Glicósidos/química , Cianuro de Hidrógeno/metabolismo , Manihot/química , Manihot/genética , Metabolómica , Estructura Molecular , Prunus/química , Prunus/genética , Sorghum/química , Sorghum/genética , Espectrometría de Masas en TándemRESUMEN
As it pertains to insect herbivores, the preference-performance hypothesis posits that females will choose oviposition sites that maximize their offspring's fitness. However, both genetic and environmental cues contribute to oviposition preference, and occasionally "oviposition mistakes" occur, where insects oviposit on hosts unsuitable for larval development. Pieris virginiensis is a pierine butterfly native to North America that regularly oviposits on an invasive plant, Alliaria petiolata, but the caterpillars are unable to survive. Alliaria petiolata has high concentrations of the glucosinolate sinigrin in its tissues, as well as a hydroxynitrile glucoside, alliarinoside. We investigated sinigrin as a possible cause of mistake oviposition, and sinigrin and alliarinoside as possible causes of larval mortality. We found that sinigrin applied to leaves of Cardamine diphylla, a major host of P. virginiensis that does not produce sinigrin, had no effect on oviposition rates. We tested the effect of sinigrin on larval performance using two host plants, one lacking sinigrin (C. diphylla) and one with sinigrin naturally present (Brassica juncea). We found no effect of sinigrin application on survival of caterpillars fed C. diphylla, but sinigrin delayed pupation and decreased pupal weight. On B. juncea, sinigrin decreased survival, consumption, and caterpillar growth. We also tested the response of P. virginiensis caterpillars to alliarinoside, a compound unique to A. petiolata, which was applied to B. oleracea. We found a significant reduction in survival, leaf consumption, and caterpillar size when alliarinoside was consumed. The 'novel weapon' alliarinoside likely is largely responsible for larval failure on the novel host A. petiolata. Sinigrin most likely contributes to the larval mortality observed, however, we did not observe any effect of sinigrin on oviposition by P. virginiensis females. Further research needs to be done on non-glucosinolate contact cues, and volatile signals that may induce P. virginiensis oviposition.
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Brassicaceae/química , Mariposas Diurnas/efectos de los fármacos , Cadena Alimentaria , Glucósidos/farmacología , Glucosinolatos/farmacología , Nitrilos/farmacología , Oviposición/efectos de los fármacos , Animales , Mariposas Diurnas/crecimiento & desarrollo , Mariposas Diurnas/fisiología , Cardamine/química , Femenino , Especies Introducidas , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/fisiología , Longevidad/efectos de los fármacos , Planta de la Mostaza/química , New York , Hojas de la Planta/químicaRESUMEN
In comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific ß-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a ß-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.
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Sistema Enzimático del Citocromo P-450/metabolismo , Glucósidos/metabolismo , Lotus/metabolismo , Manihot/metabolismo , Sorghum/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/genética , Genes Reporteros , Glucósidos/química , Hidrólisis , Lotus/química , Lotus/citología , Lotus/genética , Manihot/química , Manihot/citología , Espectrometría de Masas , Mutación , Nitrilos/química , Nitrilos/metabolismo , Hojas de la Planta/química , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Tubérculos de la Planta/química , Tubérculos de la Planta/citología , Tubérculos de la Planta/metabolismo , Regiones Promotoras Genéticas/genética , Plantones/química , Plantones/citología , Plantones/metabolismo , Sorghum/química , Espectrometría de Masa por Ionización de Electrospray/instrumentación , beta-Glucosidasa/metabolismoRESUMEN
Covering: up to the end of 2013 New mass spectrometry imaging (MSI) techniques are gaining importance in the analysis of plant metabolite distributions, and significant technological improvements have been introduced in the past decade. This review provides an introduction to the different MSI techniques and their applications in plant science. The most common methods for sample preparation are described, and the review also features a comprehensive table of published studies in MSI of plant material. A number of significant works are highlighted for their contributions to advance the understanding of plant biology through applications of plant metabolite imaging. Particular attention is given to the possibility for imaging of surface metabolites since this is highly dependent on the methods and techniques which are applied in imaging studies.
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Productos Biológicos/química , Espectrometría de Masas/métodos , Plantas/metabolismo , Productos Biológicos/análisis , Estructura Molecular , Plantas/químicaRESUMEN
Specialized metabolites in plants influence their interactions with other species, including herbivorous insects, which may adapt to tolerate defensive phytochemicals. The chemical arsenal of Alliaria petiolata (garlic mustard, Brassicaceae) includes the glucosinolate sinigrin and alliarinoside, a hydroxynitrile glucoside with defensive properties to glucosinolate-adapted specialists. To further our understanding of the chemical ecology of A. petiolata, which is spreading invasively in North America, we investigated the metabolite profile and here report a novel natural product, petiolatamide, which is structurally related to sinigrin. In an extensive study of North American populations of A. petiolata, we demonstrate that genetic population differences as well as developmental regulation contribute to variation in the leaf content of petiolatamide, alliarinoside, sinigrin, and a related glycoside. We furthermore demonstrate widely different metabolic fates of these metabolites after ingestion in the glucosinolate-adapted herbivore Pieris rapae, ranging from simple passage over metabolic conversion to sequestration. The differences in metabolic fate were influenced by plant ß-glucosidases, insect-mediated degradation, and the specificity of the larval gut transport system mediating sequestration.
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Brassicaceae/fisiología , Mariposas Diurnas/fisiología , Glucósidos/metabolismo , Glucosinolatos/metabolismo , Herbivoria , Nitrilos/metabolismo , Animales , Brassicaceae/química , Glucósidos/análisis , Glucosinolatos/análisis , Nitrilos/análisis , Hojas de la Planta/química , Hojas de la Planta/fisiologíaRESUMEN
Seed germination is crucial for plant productivity, and the biochemical changes during germination affect seedling survival, plant health and yield. While the general metabolism of germination is extensively studied, the role of specialized metabolism is less investigated. We therefore analyzed the metabolism of the defense compound dhurrin during sorghum (Sorghum bicolor) grain germination and early seedling development. Dhurrin is a cyanogenic glucoside, which is catabolized into different bioactive compounds at other stages of plant development, but its fate and role during germination is unknown. We dissected sorghum grain into three different tissues and investigated dhurrin biosynthesis and catabolism at the transcriptomic, metabolomic and biochemical level. We further analyzed transcriptional signature differences of cyanogenic glucoside metabolism between sorghum and barley (Hordeum vulgare), which produces similar specialized metabolites. We found that dhurrin is de novo biosynthesized and catabolized in the growing embryonic axis as well as the scutellum and aleurone layer, two tissues otherwise mainly acknowledged for their involvement in release and transport of general metabolites from the endosperm to the embryonic axis. In contrast, genes encoding cyanogenic glucoside biosynthesis in barley are exclusively expressed in the embryonic axis. Glutathione transferase enzymes (GSTs) are involved in dhurrin catabolism and the tissue-resolved analysis of GST expression identified new pathway candidate genes and conserved GSTs as potentially important in cereal germination. Our study demonstrates a highly dynamic tissue- and species-specific specialized metabolism during cereal grain germination, highlighting the importance of tissue-resolved analyses and identification of specific roles of specialized metabolites in fundamental plant processes.
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Grano Comestible , Sorghum , Grano Comestible/genética , Sorghum/genética , Sorghum/metabolismo , Transcriptoma , Metabolómica , Glucósidos/metabolismoRESUMEN
Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.
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Oat (Avena sativa) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum. The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea-inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea-treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea-treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.
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Monoterpenoid indole alkaloids (MIAs) are a large group of biosynthetic compounds, which have pharmacological properties. One of these MIAs, reserpine, was discovered in the 1950s and has shown properties as an anti-hypertension and anti-microbial agent. Reserpine was found to be produced in various plant species within the genus of Rauvolfia. However, even though its presence is well known, it is still unknown in which tissues Rauvolfia produce reserpine and where the individual steps in the biosynthetic pathway take place. In this study, we explore how matrix assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometry imaging (MSI) can be used in the investigation of a proposed biosynthetic pathway by localizing reserpine and the theoretical intermediates of it. The results show that ions corresponding to intermediates of reserpine were localized in several of the major parts of Rauvolfia tetraphylla when analyzed by MALDI- and DESI-MSI. In stem tissue, reserpine and many of the intermediates were found compartmentalized in the xylem. For most samples, reserpine itself was mainly found in the outer layers of the sample, suggesting it may function as a defense compound. To further confirm the place of the different metabolites in the reserpine biosynthetic pathway, roots and leaves of R. tetraphylla were fed a stable-isotope labelled version of the precursor tryptamine. Subsequently, several of the proposed intermediates were detected in the normal version as well as in the isotope labelled versions, confirming that they were synthesized in planta from tryptamine. In this experiment, a potential novel dimeric MIA was discovered in leaf tissue of R. tetraphylla. The study constitutes to date the most comprehensive spatial mapping of metabolites in the R. tetraphylla plant. In addition, the article also contains new illustrations of the anatomy of R. tetraphylla.
Asunto(s)
Rauwolfia , Alcaloides de Triptamina Secologanina , Alcaloides de Triptamina Secologanina/química , Rauwolfia/metabolismo , Reserpina/química , Reserpina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triptaminas/metabolismo , Antihipertensivos , Alcaloides Indólicos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Sequestration of plant secondary metabolites is a widespread phenomenon among aposematic insects. Sarmentosin is an unsaturated γ-hydroxynitrile glucoside known from plants and some Lepidoptera. It is structurally and biosynthetically closely related to cyanogenic glucosides, which are commonly sequestered from food plants and/or de novo synthesized by lepidopteran species. Sarmentosin was found previously in Parnassius (Papilionidae) butterflies, but it was not known how the occurrence was related to food plants or whether Parnassius species could biosynthesize the compound. Here, we report on the occurrence of sarmentosin and related compounds in four different Parnassius species belonging to two different clades, as well as their known and suspected food plants. There were dramatic differences between the two clades, with P. apollo and P. smintheus from the Apollo group containing high amounts of sarmentosin, and P. clodius and P. mnemosyne from the Mnemosyne group containing low or no detectable amounts. This was reflected in the larval food plants; P. apollo and P. smintheus larvae feed on Sedum species (Crassulaceae), which all contained considerable amounts of sarmentosin, while the known food plants of the two other species, Dicentra and Corydalis (Fumariaceae), had no detectable levels of sarmentosin. All insects and plants containing sarmentosin also contained other biosynthetically related hydroxynitrile glucosides in patterns previously reported for plants, but not for insects. Not all findings could be explained by sequestration alone and we therefore hypothesize that Parnassius species are able to de novo synthesize sarmentosin.