In Vivo31P and Multilabel13C NMR Measurements for Evaluation of Plant Metabolic Pathways

Rijhwani, Sushil K.; Ho, Chih-Huang; Shanks, Jacqueline V.

doi:10.1006/mben.1998.0102

Cited by 11 publications

(10 citation statements)

References 44 publications

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“…The identification of cytoplasmic and vacuolar Pi was verified from standard curves of 31 P chemical shift with pH in B5-F medium and in an intracellular-like synthetic medium (Fig. 9), as described by Rijhwani et al (1999). Cytoplasmic and vacuolar pH were estimated at around 7.5 and 5.5, respectively, while comparing 31 P chemical shifts of 2.77 ppm and 0.78 ppm in intracellular-like synthetic medium and the literature.…”

Section: Perfused Bioreactor Allows For In Vivo 31 P Nmr Spectroscopymentioning

confidence: 68%

“…Perfusion medium mixing and oxygenation were provided by an air flow rate of 1500 mL/min through a porous (2 Am) stainless steel sparger. The global design of the bioreactor was based on that described by Ratcliffe et al (2001), Aubert et al (1996), and Rijhwani et al (1999). A dissolved oxygen (DO) probe (polarographic: Ingold, Urdorf, Switzerland, Cat.…”

Section: Description Of the Bioreactor Systemmentioning

confidence: 99%

“…A hollow-fiber bioreactor has been developed to perform 31 P NMR on chick chondrocytes (Petersen et al, 2000). For plant cells, intracellular pH differences between the cytoplasm (pH f7.5) and the vacuole (pH f5.5) are used to quantify nutrient and metabolite compartmentalization (Bligny and Douce, 2002;Aubert et al, 1996;Roby et al, 1988;Rijhwani et al, 1999). Diverse bioreactor configurations have thus been developed to perform in vivo NMR.…”

Section: Introductionmentioning

confidence: 99%

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Development of a small-scale bioreactor: Application to in vivo NMR measurement

Gmati

Chen

Jolicœur

2004

Biotechnol. Bioeng.

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A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/ min. Residence time distribution assays at 0.8 -2.6 mL/ min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for 31 P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP g and ADP h , ATP a and ADP a , NADP and NDPG, NDPG and ATP h . Cell viability was shown to be maintained as 31 P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH. B 2004 Wiley Periodicals, Inc.

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Section: Perfused Bioreactor Allows For In Vivo 31 P Nmr Spectroscopymentioning

confidence: 68%

Section: Description Of the Bioreactor Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a small-scale bioreactor: Application to in vivo NMR measurement

Gmati

Chen

Jolicœur

2004

Biotechnol. Bioeng.

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“…Accumulation of malate occurred during the first three days (time of NO 3 À depletion from the culture medium) and intracellular malate concentration subsequently decreased until the 12th day as observed for C. roseus hairy roots (Rijhwani et al, 1999). It is now well established that nitrate uptake leads to a stimulation of organic acids synthesis in plants through a direct increase in phosphoenolpyruvate carboxylase (PEPc) activity (Murchie et al, 2000 and references therein).…”

Section: Growth Nutrient Accumulation and Elicitability In Cs3 Cellsmentioning

confidence: 96%

Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

Lamboursain

Jolicœur

2005

Biotech & Bioengineering

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In this study, we investigated the influence of initial internal nutrient concentrations at the time of elicitation on the ability of Eschscholzia californica (EC) cells to produce alkaloids. Three EC cell suspensions cultivated in culture media differing in their PO4(3-) and NO3- contents were sampled daily for 12 days and analyzed for extracellular and intracellular nutrient concentrations. The ability of the cells to produce alkaloids was tested along the three cell suspension cultures. Sampled cells were then further cultured for 7 days in a production medium containing the elicitor and an extraction resin. The alkaloid production of the cells was measured 7 days post-elicitation. In the low-N medium, starch, glucose, and phosphate contents in the biomass was increased by 470, 1624 and 70%, respectively, 10 days after inoculation compared to the control culture in standard B5 medium. Cell concentration was significantly reduced from 10.3 to 8.6 millions cell/mL on this low-N medium compared to the control, nevertheless alkaloid production was multiplied by 39 at day 10 when cells were elicited. Cells grown on the low-N or low-P media accumulated 83% and 188% more carbon, respectively, than control cells at the end of the culture. This intracellular C was mainly stored in the form of starch in low-P medium and both in the form of starch and glucose in the low-N medium. The ability of EC cells to produce alkaloids upon elicitation was shown to be strongly dependent on the initial intracellular C and P content at the time of elicitation. This suggests that reproducibility and productivity during EC cell culture could be enhanced by manipulating the intracellular C and P content at the time of elicitation.

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“…From both theoretical considerations [36,38] and experimental observations [6,37,48–50], a well‐suited substrate for the assessment of the forward flux within the non‐oxidative steps is [2‐ 13 C]glucose [51]. Breakdown of [2‐ 13 C]glucose via the oxidative steps of the pathway forms [1‐ 13 C]ribulose‐5‐phosphate, which, in turn, forms hexose‐phosphates labelled at C1 and/or C3 in the forward reactions (as above).…”

Section: Reversibilitymentioning

confidence: 99%

Carbohydrate cycling in micro-organisms: what can¹³C-NMR tell us?

Portais

Delort

2002

FEMS Microbiol Rev

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The extension of (13)C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as 'reversibility', 'metabolic' and 'substrate' cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the (13)C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.

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In Vivo31P and Multilabel13C NMR Measurements for Evaluation of Plant Metabolic Pathways

Cited by 11 publications

References 44 publications

Development of a small-scale bioreactor: Application to in vivo NMR measurement

Development of a small-scale bioreactor: Application to in vivo NMR measurement

Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

Carbohydrate cycling in micro-organisms: what can¹³C-NMR tell us?

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In Vivo31P and Multilabel13C NMR Measurements for Evaluation of Plant Metabolic Pathways

Cited by 11 publications

References 44 publications

Development of a small-scale bioreactor: Application to in vivo NMR measurement

Development of a small-scale bioreactor: Application to in vivo NMR measurement

Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

Carbohydrate cycling in micro-organisms: what can13C-NMR tell us?

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Product

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Carbohydrate cycling in micro-organisms: what can¹³C-NMR tell us?