Carbon isotope discrimination during dark respiration of plants :

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Carbon isotope discrimination during dark
respiration of plants

• magnitude and metabolic technical perspectives

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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)
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d13C of CO2 respired in the dark
13C-enriched (1 - 8) or 13C-depleted (1 - 4)
Compared to plant material
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Dark respiration of protoplasts
No fractionation occurs during dark
respiration of protoplasts
maize
bean
Lin Ehleringer, 1997
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Discrimination during dark respiration
Variability of metabolic fluxes may induce
variability of discrimination during dark
respiration
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Methods
d13C of respired CO2
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
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Respiratory discrimination
Respired CO2 is 13C-enriched compared to sucrose
Ghashghaie et al. (2001)
Duranceau et al. (1999)
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Answers
Questions

• 1) Does the discrimination during dark
  respiration occur?

YES
2) Does the discrimination vary among species?
YES
3) Does it vary with drought?
YES
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e d13C of respired CO2 d13C of sucrose

• Assuming sucrose as main substrate

• If another metabolite with different signature
  was used in respiration
• or

- If isotopic signature of the substrates is
variable between the cellular compartments,
Then the discrimination can not be calculated.
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Metabolic spectrum of d13C
Respired CO2 is also 13C-enriched compared to
other components of plants
Ghashghaie et al (2001)
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Discrimination - Respiration rate
Varying leaf temperature

• e ? d13C respired CO2 d13C of org. mat.

e - R
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Ordering variability
Discrimination is a linear relationship of
respiration rate
Tcherkez et al, unpublished
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Conclusions
Photosynthetic assimilation mainly of 12CO2
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
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Technical aspects of d13C-investigation of
metabolism
Second part
Max Hill Guillaume Tcherkez Claire Damesin Jaleh
Ghashghaie
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d13C of starch gt d13C of sucrose
Deléens-Provent Schwebel-Dugué (1987) Maize
Brugnoli et al (1988) Poplar cotton
Ghashghaie et al (2001) Sunflower Bean
Nicotiana
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Questions

• Does this variability come from
• - Plant material
• - Structure of starch (amylose/amylopectin)
• - Extraction methods

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Extraction methods
Freeze dried Oven dried Fresh (frozen)
Bulk organic matter
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Extraction of soluble sugars through HPLC
?
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Extraction of organic acids through HPLC (1)
example
But there are at least 3 ways to separate the
various organic acids through HPLC.
pyruvate malate a-ketoglutarate citrate fumarate s
uccinate
It depends on the acid you want to collect.
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Extraction of organic acids through HPLC (2)
Ionic exclusion
Hydrophobic partition
Perchloric acid, 40C, 1 mL/min
Sulfuric acid, 45C, 0.8 mL/min
Potassium phosphate 0.8 mL/min
pyruvic malic a-ketoglutaric citric fumaric succin
ic
Industrial compounds
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Extraction methods
Freeze dried Oven dried Fresh (frozen)
Bulk organic matter
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Comparisons of methods
Wanek et al., 2001
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Are approximations possible ?
There are weak relationships between d13C of
total soluble fraction and d13C of carbohydrates
such as sucrose and starch.
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d13C of plant cell compounds
Gleixner et al., 1993
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