Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.

The goal of this study is to map the metabolic pathways of poorly understood bacterial phytopathogen, Xanthomonas oryzae (Xoo) BXO43 fed with plant mimicking media XOM2 containing glutamate, methionine and either 40% [13C5] xylose or 40% [13C6] glucose. The metabolic networks mapped using the KEGG mapper and the mass isotopomer fragments of proteinogenic amino acids derived from GC-MS provided insights into the activities of Xoo central metabolic pathways. The average 13C in histidine, aspartate and other amino acids confirmed the activities of PPP, the TCA cycle and amino acid biosynthetic routes, respectively. The similar labelling patterns of amino acids (His, Ala, Ser, Val and Gly) from glucose and xylose feeding experiments suggests that PPP would be the main metabolic route in Xoo. Owing to the lack of annotated gene phosphoglucoisomerase in BXO43, the 13C incorporation in alanine could not be attributed to the competing pathways and hence warrants additional positional labelling experiments. The negligible presence of 13C incorporation in methionine brings into question its potential role in metabolism and pathogenicity. The extent of the average 13C labelling in several amino acids highlighted the contribution of pre-existing pools that need to be accounted for in 13C-flux analysis studies. This study provided the first qualitative insights into central carbon metabolic pathway activities in Xoo.

In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13 C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [ 13 C]glucose (99% [1-13 C]glucose or 99% [1,2-13 C]glucose or 40% [ 13 C 6 ]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13 C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3,4). This work provided evidence from 13 C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood. on July 10, 2020 by guest http://msystems.asm.org/ Downloaded from R alstonia solanacearum is one of the most destructive plant pathogens as it infects over 450 plant species (6-8), and its metabolism is poorly understood. It is a soilborne pathogen that enters plan...

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In Ralstonia solanacearum, a devastating phytopathogen whose metabolism 13 is poorly understood, we observed that Entner-Doudoroff (ED) pathway and Oxidative pentose phosphate pathway (OxPPP) bypasses glycolysis and OxPPP under 15 glucose oxidation. Evidences derived from 13 C stable isotopes feeding and genome 16 annotation based comparative metabolic network analysis supported the observations. 17 Comparative metabolic network analysis derived from the currently available 53 18 annotated R. solanacearum strains also including the recently reported strain (F1C1), 19 representing the four phylotypes confirmed the lack of key genes coding for 20 phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that 21 are relevant for glycolysis and OxPPP respectively. R. solanacearum F1C1 cells fed 22 with 13 C Glucose (99%[1-13 C]-or 99%[1,2-13 C]-or 40%[ 13 C 6 ]-glucose) followed by GC- 23MS based labelling analysis of fragments from amino acids, glycerol and ribose 24 provided clear evidence that rather than Glycolysis and OxPPP, ED pathway and Non-25 OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13 C 26 incorporation in the mass ions of alanine (m/z 260, m/z 232); valine (m/z 288, m/z 260), 27 glycine (m/z 218), serine (m/z 390, m/z 362), histidine (m/z 440, m/z 412), tyrosine (m/z 28 466, m/z 438), phenylalanine (m/z 336, m/z 308), glycerol (m/z 377) and ribose (m/z 29 160) mapped the pathways supporting the observations. The outcomes help better 30 defining the central carbon metabolic network of R. solanacearum that can be 31 integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for 32 defining the metabolic phenotypes. 33 Importance: Understanding the metabolic versatility of Ralstonia solanacearum is 37 important as it regulates the tradeoff between virulence and metabolism (1, 2) in a wide 38 range of plant hosts. Due to a lack of clear evidence until this work, several published 39 research papers reported on potential roles of Glycolysis and Oxidative pentose 40 phosphate pathways (OxPPP) in R. solanacearum (3, 4). This work provided evidence 41 from 13 C stable isotopes feeding and genome annotation based comparative metabolic 42 network analysis that Entner-Doudoroff pathway and Non-OxPPP bypasses glycolysis 43 and OxPPP during the oxidation of Glucose, one of the host xylem pool that serves as a 44 potential carbon source (5). The outcomes help better defining the central carbon 45 metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux 46 analysis as well as flux balance analysis studies for defining the metabolic phenotypes.47The study highlights the need to critically examine phytopathogens whose metabolism 48 is poorly understood. 50Introduction: Ralstonia solanacearum is one of the most destructive plant pathogen as 51 it infects over 450 plant species (6-8) and its metabolism is poorly understood. It is a 52 soil borne pathogen that enters the plants through natural openings or wounds,...

In vitro studies involving cell lines or primary cells, provide critical insights into their physiology under normal and perturbed conditions like cancer and infection. Given that there are multiple sources of carbon, nitrogen, and other nutrients available in routinely used standard media (such as DMEM, RPMI), it is vital to quantify their contribution to cellular metabolism. 13C based Isotopic tracers of the media components can be used to kinetically track their oxidation by the cell systems such as Human Lung Carcinoma (A549) cells. In this study, a universally labelled glucose tracer ([13C6]glucose) was used to quantify its metabolic contribution that provided further insights into the central carbon metabolism of A549 cells. Gas chromatography and mass spectrometry (GC-MS) based mass isotopomer analysis (average 13C) of methanolic extracts (glycerol: 5.46±3.53 % and lactate: 74.4±2.65 %), amino acids derived from acid hydrolysates of protein (Serine: 4.51±0.21 %, Glycine: 2.44±0.31 %, Alanine: 24.56±0.59 %, Glutamate: 8.81±0.85 %, Proline: 6.96±0.53 % and Aspartate: 10.72±0.95 %) and the metabolites of the culture filtrate (glycerol: 43.14±1.45 % and lactate: 81.67±0.91 %), allowed to capture the relative contribution of glucose. We observed the Warburg effect and a significant amount of lactate contributed from glucose, was released to the media. 13C glycerol of glucogenic origin was kinetically released to the culture filtrate and might be playing a critical role in metabolic reprogramming of A549 cells. Part of the protein biomass contributed from amino acids were of glucogenic origin. Besides, the workflow adopted for 13C analysis and derived average 13C of each metabolite provided a standard methodology that could be useful in defining the metabolic phenotypes of cells in normal and perturbed conditions. Understanding precisely the altered cellular metabolism to meet the biomass demand under a range of physiological conditions, kinetically, may identify pathways for targeted and effective therapeutic interventions.

Tulsi (Ocimum species), the queen of herbs is a common ingredient in beverages with perceived health benefits. Recently published Ocimum genome highlighted the presence of several genes that contributes to important phytochemicals but a comprehensive metabolite profiling to study the water soluble metabolites of Ocimum is lacking. In this study, untargeted metabolic profiling of hot water extract of fresh and air dried leaves of O. basilicum, O. sanctum and O. kilimandscharicum species employing gas chromatography and mass spectrometry (GC-MS) was attempted. Analysis of hot water extracts of Ocimum leaves will provide details of molecules consumed and species specific differences, if any. Several metabolic features including amino acids (glycine, serine glutamate), organic and other acids (succinic acid, fumaric acid, 4-amino butanoic acid), sugarsand their derivatives (glucose, sucrose, mannitol, fructose) and secondary metabolites (shikimic acid, quinic acid, catechol, gamma amino butyric acid, eugenol) were identified. Multivariate statistical analysis of GC-MS data indicated several species specific metabolic similarities and differences. Based on variable importance parameter score of >1, it was observed that in case of air dried extracts, glucose, fumaric acid, and D-mannitol displayed as important variables for species specific variation. Whereas in case of fresh leaves extracts, the variation was prominent due to xylose, D-allose and an unknown metabolic feature detected at 24 min (metabolite@24 with highest m/z 75). Phytochemical phenotype of Ocimum leaves not only shows species specific variations but these may partly explain their difference in taste and health benefits from their use as hot beverages. 2