Diapositiva 1

1
Mesophyll conductance to CO2 in Arabidopsis
thaliana
Jaume Flexas1, María Fernanda Ortuño2, Miquel
Ribas-Carbo1, Antonio Díaz-Espejo3, Igor D
Flórez-Sarasa1, Hipólito Medrano1
1Laboratori de Fisiologia Vegetal, Grup de
Recerca en Biologia de les Plantes en Condicions
Mediterrànies, Universitat de les Illes Balears.
Palma de Mallorca, Balears, Spain 2Dep. Botânica
e Engenharia Biológica. Instituto Superior de
Agronomia. Universidade Técnica de Lisboa.
Lisboa. Portugal 3Instituto de Recursos Naturales
y Agrobiología, CSIC, Apartado 1052, 41080
Sevilla, Spain

• Summary
• The close rosette growth form, short petioles
  and small leaves of Arabidopsis thaliana make
  measurements with commercial gas exchange
  cuvettes difficult. This difficulty can be
  overcome by growing Arabidopsis thaliana plants
  in ice-cream cone-like soil pots.
• This design permitted simultaneous gas exchange
  and chlorophyll fluorescence measurements from
  which the first estimates of mesophyll
  conductance to CO2 (gm) in Arabidopsis were
  obtained and used to determine photosynthetic
  limitations during plant ageing from c. 30-45
  days.
• Estimations of gm showed maximum values of 0.2
  mol CO2 m-2 s-1 bar-1, lower than expected for a
  thin-leaved annual species. The parameterization
  of the response of (AN) to chloroplast CO2
  concentrations (Cc) yielded estimations of the
  maximum velocity of carboxylation (Vc,max_Cc)
  which were also lower than those reported for
  other annual species. As A. thaliana plants aged
  from 30 to 45 days, there was a 40 decline of AN
  that was entirely the result of increased
  diffusional limitations to CO2 transfer, with gm
  being the largest.
• The results suggest that in A. thaliana AN is
  limited by low gm and low capacity for
  carboxylation. Decreased gm is the main factor
  involved in early age-induced photosynthetic
  decline.

Figure 2. The evolution of net photosynthesis
(AN) with leaf ageing (days after germination) in
plants of Arabidopsis thaliana. The values shown
are average S.E. of 6 replicates.
Table 1. Average values S.E. for the
photosynthetic parameters analyzed (n10).
Different letters indicate statistically
significant differences (P lt 0.05) between young
and old plants. The right column shows the
results of a quantitative limitation analysis
following Grassi and Magnani (2005), in which the
photosynthesis limitation in old plants is
referred to values in young plants. The analysis
solves total limitation in AN, and partial
limitations due to decreased gs (SL), gm (MCL)
and Vc,max_Cc (BL). The Ethier and Livingston
(2004) curve fitting method was used only for
young plants, while the Evans et al. (1986)
isotopic method was used only for old plants
Figure 1. Schematic drawing (A) and photograph
(B) of an Arabidopsis plant growing in substrate
overfilled pots. Rosette growing and measuring
form (A) allowed easy leaf clamping with the
chamber head of the Li-6400 (Li-Cor Inc.,
Nebraska, USA) (B) avoiding problems of leaf
fragility and also fully covering the 2 cm2 area
of the chamber with leaf tissue. The plant showed
in the picture was 30 days old.
Material and methods
. The actual photochemical efficiency of
photosystem II (fPSII) was determined by
measuring steady-state fluorescence (Fs) and
maximum fluorescence during a light-saturating
pulse of ca. 8000 mmol m-2 s-1 (Fm') following
the procedures of Genty et al. (1989). . Maximum
carboxylation capacity (Vc,max_Ci), maximum
capacity for electron transport rate (Jmax_Ci)
and the velocity for triose phosphate utilization
(VTPU_Ci) were calculated from AN-Ci curves
according to Long Bernacchi (2003). .
Estimations of gm by gas exchange and chlorophyll
fluorescence were performed by the method by
Harley et al. (1992) gm AN / (Ci - (G
(Jflu 8 (AN Rl)) / (Jflu - 4 (AN
Rl)))) were AN and Ci were taken from gas
exchange measurements at saturating light and G
and Rl were estimated using the Laisk (1977)
method. . Estimations of gm by a curve-fitting
method were performed as described by Ethier
Livingston, (2004) and Ethier et al. (2006) .
Estimation of gm by carbon isotope
discrimination. Instantaneous carbon isotope
discrimination was measured in old leaves as
described by Flexas et al., (2006). Carbon
isotope discrimination was calculated as
described by Evans et al. (1986), as D13Cobs
?(d13Co-d13Ce)/(1000 d13Co - ?(d13Co-d13Ce)),
where ? Ce / (Ce Co), and Ce and Co are the
CO2 concentrations of the air entering and
leaving the chamber, respectively. Gas-exchange
parameters AN, Ce and Co, were as determined with
the Li-6400. Because of the dual-inlet comparison
method used, the value of d13Ce was equal to 0
and d13Co was the value obtained from the isotope
analysis. Mesophyll conductance values were
determined by comparing predicted discrimination
with observed discrimination. Predicted
discrimination (Di) was calculated from the
equation by Evans et al. (1986) Dia(b-a)
pi/pa, where a is the fractionation occurring due
to diffusion in air (4.4), b is the net
fractionation by Rubisco and phosphoenolpyruvate
carboxylase (PEPC) (29) and pi and pa are the
intercellular and ambient partial pressures of
CO2 respectively. gm was calculated from the
equation (Evans von Caemmerer, 1996) Di
D13obs (29-1.8) (AN/gm)/pa, where 1.8 is the
discrimination due to dissolution and diffusion
of CO2 in water.
Acknowledgements This work was granted by project
BFU2005-03102/BFI (Plan Nacional, Spain). M.
R.-C. and A. D.-E. were beneficiaries of the
Programa Ramón y Cajal (M.E.C.). M.F.O. was
beneficiary of a Postdoctoral research fellowship
from M.E.C. We would like to thank Dr. Biel
Martorell for his technical help on the IRMS and
all the staff at the Serveis Científico-Tècnics
of the Universitat de les Illes Balears for their
help with measurements with mass
spectrometer. Meeting and travel expenses were
supported by U.I.B. and CSIC.
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