Water equilibrated at normal atmospheric pressure dissolves 11-µM CO2
forms 110-µM HCO3-
at pH 7.2 and 25 °C (Yokota and Kitaoka, 1985). While
RuBisCO fixes CO2
, phosphoenolpyruvate carboxylase (PEPC) uses HCO3-
the substrate. This characteristic confers a tremendous advantage to C4
Since the Km
of maize PEPC is as low as 20 mM (Uedan and Sugiyama,
1976), this enzyme can exhibit submaximal activity in the mesophyll cytosol.
In the case of the C4
plant maize, oxalacetate formed by PEPC in mesophyll cells
is reduced to malate and then decarboxylated by NADP+
enzyme in the mitochondria of bundle sheath cells to give rise to CO2
pyruvate (Heldt, 1997; Kanai and Edwards, 1999). Pyruvate returns to mesophyll
chloroplasts to be salvaged to phosphoenolpyruvate (PEP) by pyruvate Pi
dikinase (PPDK). The active operation of this pathway can convert HCO3-
mesophyll cytosol to CO2
concentrated in bundle sheath cells. The CO2
around RuBisCO in chloroplasts of bundle sheath cells reaches 500 µM
(von Caemmerer and Furbank, 1999), causing net CO2
fixation to be saturated at
10–15 Pa CO2
without any detectable photorespiration (Edwards and Walker,
1983). Thus, this auxiliary metabolic CO2
-pumping system confers significantly
better nitrogen investment and water-use efficiencies to C4
plants compared with
plants. If this CO2
-pumping system could be introduced into C3
transgenic plants would be expected to show highly improved photosynthetic
performance and productivity (Ku et al.
The maize PEPC gene has been introduced into rice chloroplasts (Ku et al.
1999). Although the severalfold higher PEPC activity in chloroplasts did not
influence carbon metabolism (Häusler et al.
, 2002), transgenic plants expressing
over 50 times more PEPC activity than wild type exhibited slightly higher
-fixation rates that were relatively insensitive to O2
(Ku et al.
, 1999). The
-fixation product in these transgenic plants was PGA, not C4
(Fukayama et al.
, 2000). However, the introduction of single C4
genes will not
establish a metabolic CO2
-pumping system since this transgenic rice depends on
glycolysis for the supply of PEP (Matsuoka et al.
, 2001). Maize malic enzyme
and PPDK have been individually introduced into rice plants, but positive
effects on photosynthesis have not been observed (Fukayama et al.
Tsuchida et al.
, 2001). One unexplained consequence of the ectopic expression of
the maize NADP+
-malic enzyme in C3
chloroplasts has been either the lack or
disturbance of grana, possibly indicating altered protein–protein interactions (Takeuchi et al.
, 2000). The incorporation of both PEPC and PPDK into rice,
generated by crossing of single-gene transformants, has been achieved and the
plants appeared to behave in a more C4
-like fashion (Ku et al.
, 2001). Introduction
of more than two C4
genes into C3
plants has not yet been attempted.
plants transgenic for all three genes may not fix CO2
efficiently since the diffusion of CO2
in cytosol and through membranes is rapid.
An observation that seems to support this prediction is that cyanobacteria concentrate
within cells to a level up to 103
times higher than the ambient CO2
concentration (Kaplan and Reinhold, 1999). The genes for the CO2
systems have been identified (Shibata et al.
, 2002). Endogenous carbonic anhydrase
is localized in carboxysomes where the HCO3-
is dehydrated to CO2
fixed by RuBisCO (Kaplan and Reinhold, 1999). Induction of a high level of
carbonic anhydrase activity in the cytosolic space caused conversion of HCO3-
, which was released from the cells at a rate sufficient to nullify the
pumping activity (Price and Badger, 1989). It will be important to learn more
and understand how such high local concentrations of CO2
around RuBisCO can
be maintained and possibly engineered into higher plant chloroplasts. In this
context, the C4
-type performance of Borszczowia aralocaspica
from the Gobi desert (Voznesenskaya et al.
, 2001) provides another interesting
example. In this plant, RuBisCO and NAD+
-malic enzyme are localized in chloroplasts
and mitochondria, respectively, and are located at the proximal end of
cells. Chloroplasts reside in the distal part of the cells and contain PPDK, but
not RuBisCO, while PEPC is located throughout the cell. Understanding how
such a spatial arrangement of enzymes is accomplished and maintained will be
important for the recreation of a functional C4
pathway in C3