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Carbon isotope discrimination during dark respiration of plants :

Jaleh Ghashghaie & Guillaume Tcherkez Laboratoire d’écophysiologie végétale Université Paris XI, Orsay, F RANCE. Franz Badeck (PIK-Potsdam-Germany) Eliane Deléens (IBP-Orsay-France). Carbon isotope discrimination during dark respiration of plants :.

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Carbon isotope discrimination during dark respiration of plants :

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  1. Jaleh Ghashghaie & Guillaume Tcherkez Laboratoire d’écophysiologie végétale Université Paris XI, Orsay, FRANCE Franz Badeck(PIK-Potsdam-Germany) Eliane Deléens(IBP-Orsay-France) Carbon isotope discrimination during dark respiration of plants : magnitude and metabolic & technical perspectives

  2. 12CO2 13CO2 CO2 (photo)respired Photosynthetic discrimination d13C of C3 plants is -28‰ They are depleted in 13C Carboxylation (29‰) (Photo)respiration : is it also subjected to carbon isotope discrimination ? Diffusion (4.4‰) CO2 (-8‰)

  3. d13C of CO2 respired in the dark 13C-enriched (1 - 8‰) or 13C-depleted (1 - 4‰) Compared to plant material

  4. Dark respiration of protoplasts No fractionation occurs during dark respiration of protoplasts maize bean Lin & Ehleringer, 1997

  5. Carbohydrates – glucose C-C-C-C-C-C 13C-enriched pyruvate C-C-C Other reactions CO2 PDH Other compounds such as fatty acids acetyl-CoA TCA cycle 2 CO2 Discrimination during dark respiration Variability of metabolic fluxes may induce variability of discrimination during dark respiration

  6. d13C of respired CO2 Methods 3 C3 species : Helianthus annuus Phaseolus vulgaris Nicotiana sylvestris HPLC and d13C of sucrose d13C of starch d13C of organic matter Intact leaves Plants are subjected to drought or not

  7. Respiratory discrimination Respired CO2 is 13C-enriched compared to sucrose Ghashghaie et al. (2001) Duranceau et al. (1999)

  8. Questions: Answers: YES 2) Does the discrimination vary among species? 1) Does the discrimination during dark respiration occur? YES 3) Does it vary with drought? YES

  9. e = d13C of respired CO2–d13C of sucrose • If another metabolite with different signature was used in respiration • or Assuming sucrose as main substrate - If isotopic signature of the substrates is variable between the cellular compartments, Then the discrimination can not be calculated.

  10. Metabolic spectrum of d13C Respired CO2 is also 13C-enriched compared to other components of plants Ghashghaie et al (2001)

  11. Discrimination - Respiration rate Varying leaf temperature ed13C respired CO2 –d13C of org. mat. e - R

  12. drought Increasing temperature Ordering variability Discrimination is a linear relationship of respiration rate Tcherkez et al, unpublished

  13. Photosynthetic assimilation mainly of 12CO2 Conclusions Dark respiration produces 13C-enriched CO2 Dark respiration contributes to 13C-depletion of organic matter The linear relationship between discrimination and respiratory flux has implications at the global scale

  14. Second part Technical aspects ofd13C-investigation of metabolism Max Hill Guillaume Tcherkez Claire Damesin Jaleh Ghashghaie

  15. d13C of starch > d13C of sucrose Deléens-Provent & Schwebel-Dugué (1987): Maize Brugnoli et al (1988): Poplar cotton Ghashghaie et al (2001): Sunflower Bean Nicotiana

  16. Questions Does this variability come from : - Plant material - Structure of starch (amylose/amylopectin) - Extraction methods

  17. Soluble sugars Organic acids Amino acids Soluble fraction Dowex columns HPLC Soluble sugars Extraction methods Freeze dried Oven dried Fresh (frozen) Bulk organic matter

  18. Calibration : pure compounds sucrose Fagus sylvatica extract stem Glucose Fructose First peak : Organic acids Amino acids Etc… leaves Extraction of soluble sugars through HPLC ?

  19. example Extraction of organic acids through HPLC (1) But there are at least 3 ways to separate the various organic acids through HPLC. pyruvate malate a-ketoglutarate citrate fumarate succinate It depends on the acid you want to collect.

  20. Extraction of organic acids through HPLC(2) Ionic exclusion Hydrophobic partition Perchloric acid, 40°C, 1 mL/min Sulfuric acid, 45°C, 0.8 mL/min Potassium phosphate 0.8 mL/min pyruvic malic a-ketoglutaric citric fumaric succinic Industrial compounds

  21. Soluble sugars Organic acids Amino acids Cellulose Starch Etc… Soluble fraction Non-soluble fraction Solubilization Hydrolyse (enzyme) Dowex columns HPLC Precipitation of starch Collection of glucose or maltose Soluble sugars Extraction methods Freeze dried Oven dried Fresh (frozen) Bulk organic matter

  22. Wanek et al., 2001 Comparisons of methods

  23. Are approximations possible ? There are weak relationships between d13C of total soluble fraction and d13C of carbohydrates such as sucrose and starch.

  24. d13C of plant cell compounds Gleixner et al., 1993

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