RESUMEN
The importance of metabolite modification and species-specific metabolic pathways has long been recognized. However, linking the chemical structure of metabolites to gene function in order to explore the genetic and biochemical basis of metabolism has not yet been reported in wheat (Triticum aestivum). Here, we profiled metabolic fragment enrichment in wheat leaves and consequently applied chemical-tag-based semi-annotated metabolomics in a genome-wide association study in accessions of wheat. The studies revealed that all 1,483 quantified metabolites have at least one known functional group whose modification is tailored in an enzyme-catalyzed manner and eventually allows efficient candidate gene mining. A Triticeae crop-specific flavonoid pathway and its underlying metabolic gene cluster were elucidated in further functional studies. Additionally, upon overexpressing the major effect gene of the cluster TraesCS2B01G460000 (TaOMT24), the pathway was reconstructed in rice (Oryza sativa), which lacks this pathway. The reported workflow represents an efficient and unbiased approach for gene mining using forward genetics in hexaploid wheat. The resultant candidate gene list contains vast molecular resources for decoding the genetic architecture of complex traits and identifying valuable breeding targets and will ultimately aid in achieving wheat crop improvement.
Asunto(s)
Estudio de Asociación del Genoma Completo , Triticum , Triticum/genética , Triticum/metabolismo , Metabolómica , Fenotipo , Redes y Vías Metabólicas/genéticaRESUMEN
Plants produce numerous metabolites that are important for their development and growth. However, the genetic architecture of the wheat metabolome has not been well studied. Here, utilizing a high-density genetic map, we conducted a comprehensive metabolome study via widely targeted LC-MS/MS to analyze the wheat kernel metabolism. We further combined agronomic traits and dissected the genetic relationship between metabolites and agronomic traits. In total, 1260 metabolic features were detected. Using linkage analysis, 1005 metabolic quantitative trait loci (mQTLs) were found distributed unevenly across the genome. Twenty-four candidate genes were found to modulate the levels of different metabolites, of which two were functionally annotated by in vitro analysis to be involved in the synthesis and modification of flavonoids. Combining the correlation analysis of metabolite-agronomic traits with the co-localization of methylation quantitative trait locus (mQTL) and phenotypic QTL (pQTL), genetic relationships between the metabolites and agronomic traits were uncovered. For example, a candidate was identified using correlation and co-localization analysis that may manage auxin accumulation, thereby affecting number of grains per spike (NGPS). Furthermore, metabolomics data were used to predict the performance of wheat agronomic traits, with metabolites being found that provide strong predictive power for NGPS and plant height. This study used metabolomics and association analysis to better understand the genetic basis of the wheat metabolism which will ultimately assist in wheat breeding.
Asunto(s)
Grano Comestible/metabolismo , Carácter Cuantitativo Heredable , Triticum/metabolismo , Genes de Plantas/genética , Estudios de Asociación Genética , Metabolómica , Fitomejoramiento/métodos , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Triticum/genéticaRESUMEN
Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Modelos Moleculares , Níquel/metabolismo , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ureasa/metabolismo , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/aislamiento & purificación , Apoenzimas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Células Cultivadas , Células Clonales , Secuencia Conservada , Activación Enzimática , Eliminación de Gen , Hidroponía , Mutación , Oryza/enzimología , Oryza/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Nicotiana/citología , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Ureasa/química , Ureasa/genética , Ureasa/aislamiento & purificaciónRESUMEN
Nucleotide catabolism in Arabidopsis thaliana and Saccharomyces cerevisiae leads to the release of ribose, which requires phosphorylation to ribose-5-phosphate mediated by ribokinase (RBSK). We aimed to characterize RBSK in plants and yeast, to quantify the contribution of plant nucleotide catabolism to the ribose pool, and to investigate whether ribose carbon contributes to dark stress survival of plants. We performed a phylogenetic analysis and determined the kinetic constants of plant-expressed Arabidopsis and yeast RBSKs. Using mass spectrometry, several metabolites were quantified in AtRBSK mutants and double mutants with genes of nucleoside catabolism. Additionally, the dark stress performance of several nucleotide metabolism mutants and rbsk was compared. The plant PfkB family of sugar kinases forms nine major clades likely representing distinct biochemical functions, one of them RBSK. Nucleotide catabolism is the dominant ribose source in plant metabolism and is highly induced by dark stress. However, rbsk cannot be discerned from the wild type in dark stress. Interestingly, the accumulation of guanosine in a guanosine deaminase mutant strongly enhances dark stress symptoms. Although nucleotide catabolism contributes to carbon mobilization upon darkness and is the dominant source of ribose, the contribution appears to be of minor importance for dark stress survival.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nucleótidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ribosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Oscuridad , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/efectos de la radiaciónRESUMEN
Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L-1, 85.22% higher than that of the wild type (7.51 g L-1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 µmol gDCW-1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.
Asunto(s)
1-Pirrolina-5-Carboxilato Deshidrogenasa/genética , Bacillus licheniformis/genética , Proteínas Bacterianas/genética , Ácido Poliglutámico/análogos & derivados , Especies Reactivas de Oxígeno/metabolismo , 1-Pirrolina-5-Carboxilato Deshidrogenasa/metabolismo , Bacillus licheniformis/enzimología , Proteínas Bacterianas/metabolismo , Citoplasma , Eliminación de Gen , Ácido Poliglutámico/biosíntesis , Ácido Poliglutámico/genéticaRESUMEN
Oxidative damage caused by the accumulation of reactive oxygen species (ROS) is one of the main obstacles to the improvement of microbial cell growth and fermentation characteristics under adverse environments. And the antioxidant capacity of cells will increase with the cell growth. Here, we found that a transition state transcription factor AbrB related to changes in cell growth status could regulate the accumulation of ROS and antioxidant capacity in Bacillus licheniformis. The results showed that the accumulation of intracellular ROS was reduced by 23.91â¯% and the cell survival rates were increased by 1.77-fold under 0.5â¯mM H2O2 when AbrB was knocked out. We further mapped regulatory target genes of AbrB related to ROS generation or clearance based on our previously analyzed transcriptome sequencing. It proved that AbrB could promote ROS generation via upregulating the synthesis of oxidase and siderophores, and negatively regulating the synthesis of iron chelators (pulcherriminic acid, and H2S). Additionally, AbrB could inhibit ROS clearance by negatively regulating the synthesis of antioxidase (superoxide dismutase, catalase, peroxidase, thioredoxin, thioredoxin reductase) and cysteine. Those results illustrated that the inactivation of AbrB during the stationary phase, along with its control over ROS generation and clearance, might represent a vital self-protection mechanism during cell evolution. Overall, the systematic investigation of the multi-pathway regulation network of ROS generation and clearance highlights the important function of AbrB in maintaining intracellular redox balance.
Asunto(s)
Antioxidantes , Bacillus licheniformis , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno , Especies Reactivas de Oxígeno , Factores de Transcripción , Especies Reactivas de Oxígeno/metabolismo , Bacillus licheniformis/metabolismo , Bacillus licheniformis/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Peróxido de Hidrógeno/metabolismo , Antioxidantes/metabolismo , Estrés Oxidativo , Sideróforos/metabolismoRESUMEN
Bread wheat is a staple food crop that is consumed worldwide. In this study, using widely targeted LC-MS/MS, we conducted a high-throughput metabolomic analysis and determined the contents and spatial distribution of metabolites in pearled fractions of the dried kernels of six representative bread wheat varieties cultivated in China. Our aim was to explore the cultivars and pearling fractions with a view toward developing functional food products. We accordingly identified notable differences in the nutrient and bioactive metabolomes, and established that the pearling fractions of each cultivar had distinct metabolic profiles. Flavonoids varied the most amongst the cultivars and were found in higher concentration in the outer layers of the grain, but only at low concentrations in the kernel. Data from this study add further evidence of benefits of whole grain wheat consumption but, specifically, medium-gluten and pigmented wheat offer other nutrient and bioactive benefits whole grain products.
Asunto(s)
Pan , Triticum , Pan/análisis , Cromatografía Liquida , Grano Comestible , Espectrometría de Masas en TándemRESUMEN
Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3'5'H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds.
Asunto(s)
Variación Genética , Genoma de Planta , Melaleuca/genética , Linaje , Árboles/genética , Alelos , Vías Biosintéticas/genética , Camellia sinensis/genética , Catequina/metabolismo , China , Domesticación , Evolución Molecular , Ácido Gálico/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Estudio de Asociación del Genoma Completo , Genómica , Análisis de Secuencia de ARN , Aceite de Árbol de TéRESUMEN
Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy.