Metabolite Profiling and Metabolomics of Plant Systems Using 1H NMR and GC‐MS

Shree, Manu; Lingwan, Maneesh; Masakapalli, Shyam Kumar

doi:10.1002/9781119509967.ch7

Cited by 14 publications

(8 citation statements)

References 47 publications

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“…Fifty microliters of supernatant was dried in a speed vacuum (Thermo Scientific). To obtain the trimethylsilyl (TMS) derivatives, the vacuum-dried extracts were dissolved in 35 l of freshly prepared hydrochloride (MeOX; Sigma-Aldrich) in pyridine (20 mg/ml) and incubated on a thermoshaker at 37°C at 900 rpm for 2 h. After that, 49 l of MSTFA was added, and the samples were incubated at 37°C and 900 rpm for 30 min on a thermomixer (36). The derivatized samples were centrifuged for 10 min at 13,000 rpm, and the supernatant was transferred to glass vials for GC-MS.…”

Section: Methodsmentioning

confidence: 99%

The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

et al. 2020

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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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Section: Methodsmentioning

confidence: 99%

The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

et al. 2020

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“…To obtain the TMS derivatives, the vacuum dried extracts were dissolved in 35 μl of freshly prepared methoxyamine hydrochloride (MeOX, Sigma Aldrich) in pyridine (20 mg/ml) and incubated on a thermoshaker at 37 °C, 900 rpm for 2 hrs. After that 49 μl of MSTFA was added and incubated the samples at 37 °C with, 900 rpm for 30 min on a thermomixer (36). The derivatised samples were centrifuged for 10 min at 13000 rpm and supernatant was transferred to glass vials for GC-MS.…”

Section: Methodsmentioning

confidence: 99%

Entner–Doudoroff pathway and Non-OxPPP bypasses glycolysis and OxPPP inRalstonia solanacearum

Jyoti

Shree

Joshi

et al. 2020

Preprint

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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,...

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“…The ramp rate was set for 10 °C/min and 5 °C/min with a hold time of 10 min for each to achieved 200 ℃ and 300 ℃ temperature respectively (Table S1). All the acquisition parameters were controlled by the chemstation software (Shree et al, 2019) (Saini et al, 2020).…”

Section: Gc-ms Data Acquisition Parametersmentioning

confidence: 99%

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis

Lingwan

Masakapalli

2021

Preprint

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Studies on specialised metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibits spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimised the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved by through a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment b) nitrogen gas drying and c) their derivatisation using MSTFA for GC-MS analysis. The optimised protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), hydroxycinnamic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. Notably, vanillin (253 m/z) was detected only in the UV-B exposed tissues. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems.

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Metabolite Profiling and Metabolomics of Plant Systems Using 1H NMR and GC‐MS

Cited by 14 publications

References 47 publications

The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

Entner–Doudoroff pathway and Non-OxPPP bypasses glycolysis and OxPPP inRalstonia solanacearum

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis

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