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Plant Physiol. (1998) 117: 1071-1081
Expression of Tobacco Carbonic Anhydrase in the C4
Dicot Flaveria bidentis Leads to Increased Leakiness of the
Bundle Sheath and a Defective CO2-Concentrating Mechanism
Martha Ludwig*, 1,
Susanne von Caemmerer,
G. Dean Price,
Murray R. Badger, and
Robert T. Furbank
Molecular Plant Physiology Group, Research School of Biological
Sciences, Australian National University, G.P.O. Box 475, Canberra
2601, Australia (M.L., S.v.C., G.D.P., M.R.B.); and Commonwealth
Scientific and Industrial Research Organization, Division of Plant
Industry, G.P.O. Box 1600, Canberra 2601, Australia (R.T.F.)
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ABSTRACT |
Flaveria bidentis (L.)
Kuntze, a C4 dicot, was genetically transformed with a
construct encoding the mature form of tobacco (Nicotiana
tabacum L.) carbonic anhydrase (CA) under the control of a
strong constitutive promoter. Expression of the tobacco CA was detected
in transformant whole-leaf and bundle-sheath cell (bsc) extracts by
immunoblot analysis. Whole-leaf extracts from two CA-transformed lines
demonstrated 10% to 50% more CA activity on a
ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the
extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This
increased CA activity resulted in plants with moderately reduced rates
of CO2 assimilation (A) and an appreciable increase in C
isotope discrimination compared with the controls. With increasing
O2 concentrations up to 40% (v/v), a greater inhibition of
A was found for transformants than for wild-type plants; however, the
quantum yield of photosystem II did not differ appreciably between
these two groups over the O2 levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher
O2 concentrations, the transformants had increased rates of
photorespiration. Thus, the expression of active tobacco CA in the
cytosol of F. bidentis bsc and mesophyll cells perturbed
the C4 CO2-concentrating mechanism by
increasing the permeability of the bsc to inorganic C and, thereby,
decreasing the availability of CO2 for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.
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INTRODUCTION |
The C4 photosynthetic pathway functions as a
CO2-concentrating mechanism by raising the
concentration of CO2 around Rubisco. As a result,
the carboxylase activity of the enzyme operates near its
Vmax, and the oxygenase activity and
photorespiration are suppressed. An important aspect of the
C4 pathway is that the reactions of
photosynthesis are divided between the mesophyll and the bsc; Rubisco
and the C3 photosynthetic C reduction cycle are
located exclusively in the chloroplasts of the bundle sheath, whereas
the initial assimilation of atmospheric CO2 takes
place in the mesophyll cell cytosol (Hatch, 1987 ). This partitioning of
the photosynthetic reactions between distinct cell types results in a
CO2-concentrating mechanism whereby
CO2 is pumped into the bundle sheath, reaching
levels up to 20 times higher than those of the surrounding mesophyll
cells (Jenkins et al., 1989).
The enzyme CA (EC 4.2.1.1) catalyzes the reversible interconversion of
CO2 and
HCO3 and, in higher plants,
may represent up to 2% of the soluble-leaf protein (Okabe et al.,
1984). Multiple forms of CA have been reported from leaf tissue of both
C3 and C4 plants (Atkins et
al., 1972; Reed and Graham, 1981; Fett and Coleman, 1994; Ludwig and
Burnell, 1995; Rumeau et al., 1996; Burnell and Ludwig, 1997). In
plants demonstrating C3 photosynthesis, most of
the CA activity is localized to the stroma of the mesophyll
chloroplasts (Poincelot, 1972; Jacobson et al., 1975; Tsuzuki et al.,
1985), where it is believed to facilitate the diffusion of
CO2 across the chloroplast envelope (Reed and
Graham, 1981; Cowan, 1986; Price et al., 1994). In
C4 plants, however, most of the CA activity is
found in the mesophyll cell cytosol (Gutierrez et al., 1974; Ku and
Edwards, 1975; Burnell and Hatch, 1988), where it catalyzes the
hydration of atmospheric CO2 to
HCO3, which is the substrate
for the primary carboxylating enzyme of the C4
pathway, PEP carboxylase (Hatch and Burnell, 1990).
Biochemical and modeling studies (Furbank and Hatch, 1987; Burnell and
Hatch, 1988) have previously maintained that the absence of CA in the
bundle sheath of C4 plants is essential for an
effective CO2-concentrating mechanism and,
thereby, efficient functioning of the C4 pathway.
However, when Jenkins et al. (1989) considered the presence of CA in
the bundle-sheath cytosol in their model, they predicted that even a
1000-fold increase in CA activity over the noncatalyzed rate would have
only a moderate effect on the efficiency of C4
photosynthesis.
The recent development of a transformation system for the NADP-ME-type
C4 dicot Flaveria bidentis (L.) Kuntze
(Chitty et al., 1994) offers the opportunity to genetically manipulate
various aspects of the C4 photosynthetic pathway
(Furbank et al., 1997). To directly determine the effects of elevated
CA activity in the cytosol of C4 bsc and
mesophyll cells, we have transformed wild-type F. bidentis
plants with a construct encoding tobacco (Nicotiana tabacum
L.) CA without the putative chloroplast transit peptide. Because this
sequence encoding the mature form of the tobacco enzyme was under the
control of the constitutive CaMV 35S promoter, expression of the
tobacco CA was expected in the cytosol of both the mesophyll and bsc of
the transformants. The expression of this introduced CA in F. bidentis and its activity, as well as the effects of this
expression on the efficiency of C4
photosynthesis, are described.
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MATERIALS AND METHODS |
Expression of Tobacco CA in Escherichia coli and
Generation of Antiserum
A cDNA clone encoding tobacco (Nicotiana tabacum L.) CA
(Price et al., 1994) was adapted for expression in E. coli through the use of the following PCR oligonucleotides:
5 -AGAGTTGACGGATCCATGGCTGAATTGCAATC and
5-CACTTAAAACGGAATTCGAGCTCATACGGAA. The first primer allowed the
removal of the transit peptide-coding region of tobacco CA and
introduced a BamHI site followed by an NcoI site
near the putative start of the mature peptide, which was assumed to be Gln-101 (Majeau and Coleman, 1992) by analogy to spinach CA (Burnell et
al., 1990). These modifications resulted in a start sequence of MAELQ
for the recombinant gene product. The second primer introduced SacI and EcoRI sites immediately downstream of
the tobacco CA cDNA stop codon. The resulting 660-bp PCR product was
digested with BamHI and EcoRI and cloned into the
corresponding sites of the E. coli expression vector
pTrcHisA (Invitrogen, Carlsbad, CA). The resulting construct, named
ptrcTOBCA, was further modified by the deletion of a 104-bp
NcoI/NcoI fragment, thus removing the polyHis
leader from pTrcHisA. This second construct was named ptrcTOBCA-Nco/Nco. Both constructs expressed the recombinant CA at high
levels (up to 20% of soluble protein) in E. coli JM109 cells, and the protein was fully active in the presence of DTT and had
properties identical to CA from tobacco leaf extracts (G.D. Price,
unpublished data). Protein expressed from ptrcTOBCA-Nco/Nco was
purified by SDS-PAGE and used for the generation of rabbit polyclonal
antibodies essentially as described by Price et al. (1995). To reduce
nonspecific binding, antiserum was preabsorbed to an acetone powder of
E. coli JM109 cells (Harlow and Lane, 1988).
Construction of pBI-CA-GUS Binary Plasmid
The region encoding mature tobacco CA was removed from ptrcTOBCA
as a 660-bp BamHI/SacI fragment and ligated into
the corresponding sites of the pBI-GUS-B6F binary plasmid, as detailed
by Price et al. (1995). The NcoI site behind the
BamHI site of the CA clone was selected to produce a context
that could act as a Kozak ribosome-binding site (Kozak, 1983).
Plant Transformation and Regeneration
Flaveria bidentis (L.) Kuntze plants were transformed
and regenerated using the pBI-CA-GUS construct and the
Agrobacterium tumefaciens method described by Chitty et al.
(1994). Selection of transformants was made on kanamycin-containing
medium. Measurements of neomycin phosphotransferase II and GUS activity
were made on the primary transformants following the procedures of
Chitty et al. (1994). Primary transformants were allowed to
self-fertilize and T1 seeds were collected. The
T1 seeds from six different primary transformants, 190-1, 190-3, 191-2, 191-4, 191-7, and 191-8, were sown and plants were grown in a naturally lit greenhouse (von Caemmerer
et al., 1997b).
Detection of Tobacco CA in T1 and T2
F. bidentis Plants
Leaf discs measuring 0.5 cm2 were collected
from the youngest, fully expanded leaves of both tobacco plants and the
progeny of the six F. bidentis primary transformed lines.
Leaf discs were snap frozen in liquid N2 and
stored at  80°C until use. Total leaf protein was extracted from a
leaf disc in 500 µL of 50 mM Hepes-KOH, pH 7.2, containing 1% (w/v) insoluble PVP, 1 mM EDTA, 10 mM DTT, 5 mM MgCl2, and 1 mM PMSF in a 2-mL ground-glass tissue homogenizer on ice.
An equal volume of 2× Tricine SDS-PAGE sample buffer (1× buffer is
450 mM Tris-HCl, pH 8.45, 12% [v/v] glycerol, 4%
[w/v] SDS, 0.0075% [w/v] Coomassie blue G, 0.0025% [w/v] phenol red, and 100 mM DTT) was added to the leaf extracts, which
were then boiled for 4 min and centrifuged to clarify. Equivalent
amounts of the extracts, based on equal leaf area of starting material, were separated on Tricine 10 to 20% (w/v) polyacrylamide gels (Novex
gels, AMRAD Biotech, Boronia, Victoria, Australia) and then blotted to
nitrocellulose (Schleicher & Schuell) using a semidry blotting
apparatus (NovaBlot Unit, AMRAD Biotech) with a continuous buffer
system (39 mM Gly, 48 mM Tris, 0.0375% [w/v] SDS, and 20% [v/v] methanol).
Gels were blotted at room temperature for 1.5 h at 0.54 mA
cm2. Nonspecific binding sites on the membranes
were blocked by an overnight incubation in 12.5 mM
Tris-HCl, pH 8.0, containing 137 mM NaCl, 2.7 mM KCl, and 1% (v/v) Tween 20 (TBST) with 5% (w/v) skim-milk powder. Blots were then incubated in a 1:1000 dilution of the
anti-tobacco CA antiserum in the blocking buffer at room temperature
for 2 h. Blots were washed three times in TBST and then labeled
with a 1:3000 dilution of horseradish peroxidase conjugated-donkey
anti-rabbit IgG secondary antibody (Amersham) in the above blocking
buffer. After several washes in TBST and a final wash in TBST without
detergent, immunoreactive polypeptides were detected using an enhanced
chemiluminescence western-blotting analysis system (Amersham).
Gas-Exchange and C Isotope Measurements
Gas-exchange measurements were made together with measurements of
C isotope discrimination,  , on attached, youngest, fully expanded
leaves using an open gas-exchange system described by von Caemmerer and
Evans (1991). C isotope discrimination was measured by collecting the
CO2 in the air stream leaving the leaf chamber with and without a leaf present, and then calculating the difference in
C isotope composition (von Caemmerer and Evans, 1991; von Caemmerer et
al., 1997b). Measurements were made at 2000 µmol quanta
m2 s1, a leaf
temperature of 25°C, and an ambient CO2
concentration of 350 µbar. Leaf discs (0.5 cm2)
taken from the same leaves after gas-exchange measurements were plunged
into liquid N2 and stored at 80°C for
subsequent biochemical assays. C isotope discrimination of leaf dry
matter was determined on the leaf opposite that used in gas exchange as
described by von Caemmerer et al. (1997b).
Gas-Exchange and Chl Fluorescence Measurements
Gas-exchange and Chl fluorescence measurements were made as
described by Siebke et al. (1997) with a pulse-modulated fluorometer (PAM 101, Walz, Effeltrich, Germany) attached to a portable
photosynthesis system (model 6400, Li-Cor, Lincoln, NE), which was
fitted with a special cuvette that held a polyfurcated fiber optic
connecting the different light sources and the measuring beam. Gases
were supplied from pressurized gas cylinders containing
N2 and O2 and mixed with
electrical mass-flow controllers (type 1179A, MKS Instruments, Andover,
MA) to obtain the desired concentrations. The CO2
concentration was adjusted with the Li-Cor 6400 CO2 injection system. The
O2 concentration of the analyzed gas is known to
influence the estimation of the water-vapor concentration by the
injection system, and the calibration at different
O2 concentrations was adjusted accordingly (S. von Caemmerer, unpublished data). The  PSII was
calculated according to the method of Genty et al. (1989).
Biochemical Measurements
Leaf discs collected at the time of gas-exchange measurements were
homogenized in 500 µL of extraction buffer as described above and
centrifuged to clarify. Aliquots of the extracts were used to quantify
soluble protein (Coomassie Plus reagent, Pierce) and Rubisco content
was determined by the
[14C]2-carboxy-D-arabinitol-1,5-bisphosphate-binding
assay (Butz and Sharkey, 1989; Mate et al., 1993). CA activity was
determined using a MS technique that measured the rate of
18O exchange from doubly labeled
13C18O2
to H216O (Badger and Price,
1989). These assays were performed with 1 mM inorganic C at
25°C with the other modifications noted by Price et al. (1994). Total
Chl content was determined according to the method of Porra et al.
(1989).
Isolation of Bundle-Sheath Strands
Upper, fully expanded leaves were collected, deribbed, and sliced
into 2- to 4-mm-wide strips. Approximately 0.6 g of leaf strips
was homogenized in 1 mL of extraction buffer as described above (equals
whole-leaf samples), whereas the remaining strips were used to prepare
bundle-sheath strands according to the method of Meister et al. (1996).
Proteins were extracted from bsc by homogenizing a concentrated aliquot
of bundle-sheath strands in 1 mL of extraction buffer. Aliquots of
whole-leaf and bsc extracts, clarified by centrifugation, were used to
determine CA activity, Rubisco content, and soluble-protein
concentration as described above. Separation of extracts by Tricine
SDS-PAGE and immunoblot analysis were carried out as described above,
except that samples were loaded onto the gel based on equivalent
amounts of Rubisco protein.
At the time of bundle-sheath strand isolation, an upper, fully expanded
leaf and bundle-sheath strands from each plant were frozen in liquid
N2 and stored at 80°C. These samples were
subsequently used to determine the extent of mesophyll contamination of
the bundle-sheath strand preparations using PEP carboxylase and
phosphoribulokinase as mesophyll and bsc marker enzymes, respectively
(Lunn and Furbank, 1997).
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RESULTS AND DISCUSSION |
Expression of Active Tobacco CA in Transgenic F. bidentis
A number of A. tumefaciens-mediated
transformation events using the mature tobacco CA construct (Fig.
1) gave rise to kanamycin-resistant
F. bidentis plants, which tested positive in both GUS and
neomycin phosphotransferase II assays (data not shown). Under the
regeneration and growth conditions used, however, no obvious visual
phenotype was detected in the transformants. No further data were
collected from the primary transformants and they were allowed to
self-fertilize and set seed. The T1 seeds from
six different primary transformants were sown and, again, the resulting
plants showed no differences in phenotype when compared with wild-type
F. bidentis plants. To determine if the
T1 progeny were expressing the mature tobacco CA
polypeptide, a large number of these plants were screened using
immunoblot analysis.
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| Figure 1.
Map of the binary construct, pBI-CA-GUS, used in
the transformation of F. bidentis. The 660-bp
BamHI/SacI fragment encoding the mature
tobacco CA polypeptide and the gus gene were flanked by
the CaMV 35S promoter and the 3 end of the nopaline synthase (nos) gene. The nptII gene was flanked by
the promoter and the 3 end of the nos gene. Direction
of transcription of the genes is indicated by the arrows.
kanr, Kanamycin resistance; RB and LB, right and left
borders of the T-DNA, respectively.
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The antiserum raised against the recombinant tobacco CA labeled the
mature form of the CA polypeptide (24 kD; Majeau and Coleman, 1992) in
tobacco leaf extracts (Fig. 2, lanes
TOB). The upper, less intensely labeled immunoreactive polypeptides
detected in the tobacco leaf extracts are probably either processing
intermediate forms of the enzyme or perhaps other isoforms of tobacco
CA, as has been suggested for pea CA (Majeau and Coleman, 1991;
Johansson and Forsman, 1992; Provart et al., 1993). The anti-tobacco CA antiserum labeled four polypeptides in control F. bidentis
leaf extracts with molecular masses of approximately 35, 32, 30, and 27.5 kD (Fig. 2, line 191-2). Previously, two distinct CA polypeptides were identified in F. bidentis leaf extracts using an
anti-spinach CA antiserum (Ludwig and Burnell, 1995). The discrepancy
between these CA-labeling patterns is likely due to differing
specificities and titers of the two antisera. Results from
N-terminal amino acid sequencing indicate that the four
F. bidentis polypeptides labeled with the anti-tobacco CA
antiserum represent different isoforms of CA (M. Ludwig, unpublished
data). Accumulating molecular evidence also indicates that CA is
encoded by a multigene family in higher plants (Fett and Coleman, 1994;
Ludwig and Burnell, 1995; Rumeau et al., 1996; Burnell and Ludwig,
1997).
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| Figure 2.
Detection of tobacco CA expression in
T1 leaf extracts using immunoblot analysis. Numbers above
the brackets designate F. bidentis primary transformed
lines, whereas the numbers directly above the gel lanes represent
individual progeny from the primary transformants. Equivalent amounts
of tobacco (TOB) and F. bidentis leaf extracts based on
equal leaf-area starting material were separated by SDS-PAGE, blotted
to nitrocellulose, and labeled with the anti-tobacco CA antiserum.
Immunoreactive polypeptides were detected with an HRP-conjugated
secondary antibody and the enhanced chemiluminescence labeling method
as described in ``Materials and Methods''. The position of the 30-kD
molecular mass marker is indicated.
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Figure 2 also shows some of the results obtained from screening
T1 individuals for the expression of tobacco CA.
Clearly, the tobacco CA polypeptide was detected in a number of progeny from several of the primary transformed lines (Fig. 2, lines 191-7, 191-4, and 191-8). However, the labeling intensity of the tobacco CA
polypeptide always appeared much less than the total immunoreactive signal from the F. bidentis CA polypeptides in these
transformants. Furthermore, the level of tobacco CA expression varied
among the lines and even among the progeny from a given primary
transformant (Fig. 2, 191-7.5 versus 191-7.8).
To determine if the tobacco CA, which was modestly expressed in the
F. bidentis transformants, was biochemically active in these
plants, a sensitive MS assay, which measured the rate of exchange of
18O from doubly labeled
13C18O2
to H216O (Badger and Price,
1989; Price et al., 1994), was used. Individuals from line 191-7, a
transformed line producing progeny expressing relatively high levels of
tobacco CA (Fig. 2), were chosen for these assays (Table
I), whereas progeny from line 191-2 were used as transformed negative controls, since the tobacco CA polypeptide was never detected in leaf extracts from any progeny of this line. Soluble-protein and Rubisco content were also measured in leaf extracts
from these plants (Table I). On a leaf-area basis, extracts from
F. bidentis plants expressing the tobacco CA demonstrated 6 to +60% more CA activity than the negative control plant extracts. However, when Rubisco and soluble-protein content were compared, no
appreciable difference was detected between the CA transformants and
the controls, nor were any differences in Chl content evident (Table
I).
Expression of Tobacco CA in the bsc of F. bidentis CA Transformants
In C4 plants most of the CA is located in
the cytosol of the mesophyll cells (Gutierrez et al., 1974; Ku and
Edwards, 1975; Burnell and Hatch, 1988), where it catalyzes the
hydration of atmospheric CO2 entering the leaf to
HCO3, which is the substrate
for the primary carboxylating enzyme of the C4
photosynthetic pathway, PEP carboxylase (Hatch and Burnell, 1990). Very
little endogenous CA has been found in the bsc of C4 plants (Burnell and Hatch, 1988), and it has
been argued that it is this characteristic absence of CA in the bundle
sheath that allows efficient functioning of the
C4 pathway (Burnell and Hatch, 1988).
Because the pBI-CA-GUS construct used in the above transformation
experiments contained the sequence encoding only the mature form of
tobacco CA (i.e. no chloroplast transit peptide sequence was present),
and because the CA sequence was under the control of the constitutive
CaMV 35S promoter, it was expected that in the transformants the
tobacco CA would be expressed in the cytosol of both mesophyll and bsc.
To determine whether this was the case, bundle-sheath strands were
isolated from plants 191-7.7 and 191-7.8 and from two transformed
plants not expressing tobacco CA, 191-2.1 and 191-2.4. Figure
3 and Table I show the results of these
experiments. The tobacco CA polypeptide was detected only in the
progeny of line 191-7; labeling of the tobacco polypeptide was clearly
seen in both the whole-leaf and bsc extracts of plants 191-7.7 and 191-7.8 (Fig. 3). When CA activity was measured on a Rubisco-site basis using aliquots of these extracts, whole-leaf samples from the CA
transformants showed a 10% to 50% increase in enzyme activity compared with the negative control plants (Table I). A more striking difference in CA activity was observed between the bsc samples of the
controls and the CA transformants; 3 to 5 times more CA activity was
measured in the bsc extracts of the CA transformants than in the
control samples (Table I).
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| Figure 3.
Detection of tobacco CA in whole-leaf and bsc
extracts of F. bidentis CA transformants. Whole-leaf
(WL) and bsc (BSC) extracts from two transformed, negative control
F. bidentis plants, 191-2.1 and 191-2.4, and two
transformants expressing tobacco CA (TOB CA), 191-7.7 and 191-7.8,
were separated by SDS-PAGE and immunolabeled with the anti-tobacco CA
antiserum. Equivalent amounts of all of the extracts based on Rubisco
content were loaded onto the gels. The position of the 30-kD molecular
mass marker is indicated. TOB, Tobacco leaf extract.
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The CA activity values reported for the bsc extracts in Table I were
corrected for mesophyll contamination using PEP carboxylase as the
mesophyll marker enzyme. A maximum mesophyll contamination of 3.5% was
estimated for all control and CA transformant bsc preparations.
Because this contamination was consistently low in all of the samples,
it may reflect the activity of the nonphotosynthetic isoform(s) of PEP
carboxylase found in the leaf tissue of Flaveria species
(Hermans and Westhoff, 1990).
It is also interesting to note that several of the F. bidentis CA polypeptides were detected in bsc extracts from
control plants (Fig. 3). Values of bsc CA activity corrected for
mesophyll contamination also suggested that significant amounts of
endogenous CA, about 5% of total leaf CA activity, are normally
present in the bundle sheath of F. bidentis (Table I).
Burnell and Hatch (1988) determined the amount of bsc CA activity for a
number of plants representing the three types of
C4-decarboxylation pathways. Values averaging
0.65% of total leaf CA activity were found for PEP carboxykinase-type
and NAD-ME-type C4 plants, whereas higher estimates of about 1.7% were reported for the NADP-ME-type plants sorghum and maize. F. bidentis is also an NADP-ME-type
C4 plant, however, the bsc CA activity determined
in the present study is appreciably higher than that of sorghum or
maize. The reason(s) for this difference is not immediately apparent
and although it may simply be due to a difference in isolation
procedures or measurement of enzyme activity, it might reflect a real
difference between NADP-ME monocots and dicots, as has been noted for
assimilation rates and the ratio of intercellular-to-ambient
CO2 partial pressure (Henderson et al., 1992).
Both the inter- and intracellular locations of CA in F. bidentis are currently under investigation.
bsc Leakiness Is Increased in the F. bidentis CA
Transformants
As mentioned above, both the primary pBI-CA-GUS transformants and
their progeny showed no differences in phenotype relative to control
plants under the growth conditions employed. However, a physiological
difference was expected at the cellular level from introducing the
tobacco CA into the bsc cytosol of F. bidentis. The diagrams
in Figure 4 show the flow of
photosynthetic C in F. bidentis wild-type plants and CA
transformants. After malate was decarboxylated in the bsc chloroplasts
of wild-type F. bidentis plants (Fig. 4A), much of the
released CO2 was fixed by Rubisco. The
CO2 that was not fixed diffused out of the
chloroplast and back into the mesophyll cells. Very little of this
CO2 was converted to
HCO3 because relatively low
amounts of CA were present in the bsc cytosol (see discussion above).
In contrast, the biochemical data presented above indicated that a
significant proportion of the tobacco CA was located and active in the
bsc of the CA transformants. Thus, in the bsc chloroplasts of these
plants (Fig. 4B), the unfixed CO2 released from
the decarboxylation of malate diffused out of the chloroplast into the
bsc cytosol, where it could then be converted more rapidly to
HCO3 due to the introduction
of tobacco CA in this compartment. An increase in bsc leakiness is
proposed to accompany this shift toward thermodynamic equilibrium,
since both CO2 and
HCO3 could then diffuse back
into the mesophyll cells of the CA transformants.
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| Figure 4.
Schematic representation of photosynthetic C flow
in F. bidentis wild-type plants (A) and CA transformants
(B). In F. bidentis, an NADP-ME-type C4
plant, CO2 is released from the C4 acid malate in the bundle-sheath chloroplasts. The two schemes differ in the predicted relative amounts of CO2 and
HCO3, which diffuse from the bundle sheath
into the mesophyll. The dashed arrows in A denote that CO2
is the major form of inorganic C diffusing back into the mesophyll in
wild-type F. bidentis plants. Whereas in the CA
transformants (B), inorganic C is lost from the bundle sheath in the
form of both CO2 and HCO3 (solid
arrows) because of the activity of mature tobacco CA
(CATOB) in this compartment. See text for further details.
C3-P, Triose-P; and RUBP, ribulose-1,5-bisphosphate.
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To determine whether the bsc of the CA transformants were more leaky to
inorganic C, these plants and the transformed negative controls were
characterized using gas-exchange and C isotope discrimination methods.
The data in Table II show that the CA
transformants demonstrated a 13% to 25% reduction in the C net
assimilation rate compared with control plants. However, the similar
values of Rubisco content (Table I) and the ratio of
intercellular-to-ambient CO2 partial pressure
(Table II) obtained for both the controls and the CA transformants show
that the decrease in the net assimilation rate was not a result of
decreased stomatal conductance or lack of carboxylating enzyme in the
CA transformants. Instead, the data are consistent with a reduction in
the concentration of CO2 in the bsc being
responsible for the observed phenotype.
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Table II.
Gas-exchange and C isotope discrimination
properties of leaves from F. bidentis control plants and CA
transformants
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The C isotope discrimination data showed that the CA transformants
exhibited appreciable increases in C isotope discrimination () when
was measured both in the short term during gas exchange and on leaf
dry matter (Table II). Figure 5 shows the
relationship between measured during gas exchange and the ratio of
intercellular-to-ambient partial pressure of CO2
(pi/pa) for
control and CA transformants. We have used the model of Farquhar (1983)
to interpret the data. The model predicts that:
where a (4.4 ) is the fractionation during diffusion
of CO2 in air (Farquhar et al., 1989).
b4 (5.7) is the combined fractionation of PEP carboxylation (2.2) and the preceding isotopic equilibrium during dissolution of CO2 and conversion to
HCO3, and assumes that
CO2 and
HCO3 are close to equilibrium
in the mesophyll cytosol. (If this were not the case, then
b4 = [5.7 + 7.9Vp/Vh],
where Vp and Vh
are the rates of PEP carboxylation and CO2
hydration, respectively.) b3 (30) is the
fractionation by Rubisco and s is the fractionation during
leakage of inorganic C out of the bundle sheath. In the absence of CA
in the bundle-sheath cytosol it is usually assumed that s is
equal to the fractionation occurring as CO2
dissolves in the bundle sheath and diffuses back to the mesophyll
(s = 1.8). When CA is present in the bundle-sheath
cytosol, however, HCO3 can
also diffuse out of the bundle sheath. Since the heavier isotope,
13C, concentrates in
HCO3 and Rubisco fixes
12C preferentially, the isotope fractionation
associated with leakage increases. Under these conditions
s = (5.3 /[1 + 5.3])(0.4 10.1) + 1.8. Thus, depending on the amount of CA in the bundle sheath, the
fractionation factor, s, associated with leakage may vary
from 1.8, when there is no
HCO3 diffusion ( = 0), to
6.3, when there is complete equilibrium between
CO2 and
HCO3 in the bundle-sheath
cytosol ( = 1) (Farquhar, 1983; von Caemmerer et al., 1997a).
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| Figure 5.
Short-term C isotope discrimination ( ) as a
function of the intercellular-to-ambient partial pressure of
CO2
(pi/pa) in
transformed negative control plants (191-2.1,  ; 191-2.5,  ; and
191-2.4,  ) and F. bidentis plants expressing tobacco
CA (191-7.6,  ; 191-7.8,  ; and 191-7.7, ). The lines are not
a fit to the data but depict the theoretically predicted relationship
where: = 4.4 + ([30 s] 10.1)pi/pa.
Slope of line (1) = 3.79; of line (2) = 0.512; and of line (3) = 0.898.
|
|
The control plants have C isotope discrimination values similar to
those measured previously for F. bidentis (Henderson et al.,
1992; von Caemmerer et al., 1997a) and a leakiness value of about 22%
is also similar when it is estimated with
b4 = 5.7 and s = 1.8. It is usually assumed that CA is present in sufficient excess
in the mesophyll cytosol of C4 species such that
b4 = 5.7, however, this topic deserves
further consideration. For example, Hatch and Burnell (1990) estimated
that the rate of CO2 hydration was between 5 and
15 times that of CO2 fixation at ambient
CO2 concentrations. Our measurements (Tables I
and II) show the CO2 hydration rate to be
approximately 5 times that of CO2 fixation for
F. bidentis, although this may be an underestimate, since loss of activity of CA may occur during extraction procedures.
The large increase in the C isotope discrimination in the CA
transformants indicates that the bsc of these plants are more leaky to
inorganic C than control bsc (Table II; Fig. 5). We calculate that the
leakiness in the CA transformants may have increased on average to
between 28 (s = 6.3) and 37%(s = 1.8).
Studies by Dai et al. (1996) have demonstrated that at atmospheric
CO2 concentrations, maximal rates of
photosynthesis are observed in F. bidentis when
O2 concentrations are between 5% and 10%.
Inhibition of photosynthesis due to photorespiration occurs at
O2 concentrations above this optimum (Dai et al.,
1996; Fig. 6A). Due to the increased
leakiness of the CA transformant bundle sheath, it was suspected that
these plants would show an increased sensitivity to
O2 compared with control F. bidentis plants. Plants from the T2 generation of CA
transformants were screened by immunoblot analysis for the expression
of tobacco CA. The CO2-assimilation rate, the
PSII, and the ratio of
PSII to A (a measure of the energy required
per CO2 molecule fixed) were examined in three
individuals exhibiting levels of tobacco CA expression similar to the
T1 plants used in the previous experiments (Fig.
6). These characteristics were also determined for three wild-type
plants. At the optimal O2 concentration, the CA
transformants showed little difference in
CO2-assimilation rates relative to wild-type
plants (Fig. 6). However, at higher O2
concentrations, the inhibition of photosynthesis was clearly more
severe in the transformants than in the wild-type plants (Fig. 6). In
contrast, at O2 concentrations above 10%,
PSII remained relatively constant for both
wild-type plants and the CA transformants (Fig. 6). Consequently, at
these O2 levels, CO2
assimilation requires more PSII activity in the CA transformants than
in wild-type F. bidentis plants (Fig. 6). These results
support the conclusion from the gas-exchange and C isotope
discrimination measurements that the bsc of the CA transformants are
more leaky to inorganic C than control bsc. Thus, as the partial
pressure of O2 increases in the bundle sheath of the CA
transformants, the oxygenation reaction of Rubisco is favored to a
greater extent than in wild-type plants. This results in the
transformants exhibiting higher photorespiration rates and decreased
CO2-assimilation rates relative to the wild-type plants at atmospheric O2 concentrations and
above.
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| Figure 6.
O2 dependence of A, PSII, and the
ratio of PSII to A in F. bidentis wild-type plants
(black symbols) and transgenic plants expressing tobacco CA (white
symbols; plants were selected from the T2 progeny of
transformant 191-7.8). Measurements were made at an ambient
CO2 concentration of 360 µbar, an irradiance of 1000 µmol quanta m2 s1, and a leaf temperature
of 25°C. Values are expressed as a percentage of values at 2%
O2. Mean values at 2% O2 were: A, 32.1 ± 1.3 and 25.8 ± 1.4; PSII, 0.53 ± 0.02 and 0.502 ± 0.01; and PSII/A, 7.2 ± 0.2 and 8.63 ± 0.2 for F. bidentis wild-type plants and transformants,
respectively.
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|
Recent studies by Siebke et al. (1997) and Maroco et al. (1998) also
examined the O2 sensitivity and
PSII of C4
photosynthesis to further characterize plants defective in the
C4 cycle, namely PEP carboxylase mutants of
Amaranthus edulis and plants with a faulty
C3 pathway (i.e. antisense small subunit Rubisco
F. bidentis plants). The results of these recent studies,
along with those reported here for the CA transformants, indicate that
this information is valuable in understanding the interactions of the
C3 and C4 cycles in the
C4 photosynthetic pathway and the effects on the pathway when the balance between the two interconnected cycles is
perturbed.
CA Transformants Contain Defective
CO2-Concentrating Mechanisms
Early work (Furbank and Hatch, 1987; Burnell and Hatch, 1988)
pointed out that the presence of CA in the bundle sheath would lead to
inefficient functioning of the C4 pathway. The
curves shown in Figure 7 were generated
using a mathematical model of C4 photosynthesis
(Berry and Farquhar, 1978; von Caemmerer et al., 1997b) and predict
what quantitative changes one might expect to observe in
CO2-assimilation rate, , and bundle-sheath
leakiness and CO2 concentration with respect to
decreasing ratios of CO2 to
HCO3 in the bundle-sheath
cytosol.
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| Figure 7.
Predicted changes in the
CO2-assimilation rate, bundle-sheath leakiness () and
CO2 partial pressure (ps), and C isotope
discrimination, with changes in the degree of CO2 and
HCO3 equilibration in the bundle-sheath
cytosol ( = 1 at full equilibration between CO2 and
HCO3). Calculations were made with the
mathematical model of C4 photosynthesis described by von
Caemmerer et al. (1997b) at an intercellular CO2 partial
pressure of 150 µbar, a maximal Rubisco activity of 50 µmol
m2 s1, a maximal PEP carboxylase activity
of 72 µmol m2 s1, and a bundle-sheath
conductance (gs) of 2 (1 + 5.3 ) mmol
m2 s1. C isotope discrimination was
calculated from = 4.4 + ([30 s] 10.1)pi/pa, with
pi/pa = 0.45 and s = 1.8 10.1(5.32/[1 + 5.3]).
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|
Farquhar (1983) considered a simplified system with uniform pH where
interconversion of CO2 and
HCO3 takes place in the
mesophyll and bsc but not in the symplastic transport path. He showed
that the bundle-sheath conductance to leakage of total inorganic C
could be given by:
where gsc is the bundle-sheath
conductance to CO2 diffusion alone and the
diffusivity of HCO3 in water,
Db, is 0.56 times that of
CO2, Dc, at 25°C
(Kigoshi and Hashitani, 1963). The dissociation constant K
for HCO3 is
106.12 at 25°C and an ionic strength of 0.1 (Harned and Bonner, 1945). Therefore, at a cytoplasmic pH of 7.4, gs = gsc (1 + 10.67
lc/lb). Jenkins
et al. (1989) inferred from metabolite transport measurements that the
effective path length for HCO3 diffusion,
lb, is approximately twice that of
CO2, lc. Thus a 6- to
7-fold increase in bundle-sheath conductance is seen as varies from
0 to 1. This increase in conductance is reflected in the decrease in A
and bundle-sheath CO2 partial pressure and in the
increase in bsc leakiness and C isotope discrimination values. With the
chosen parameters the model predicts that even at complete equilibrium
of HCO3 and
CO2 in the bsc cytosol, the
CO2-assimilation rate would be decreased by only
30%, which would be accompanied by a 2.5-fold increase in C isotope
discrimination. In line with these predictions, our data show that the
modest reduction in CO2-assimilation rate demonstrated by the CA transformants was accompanied by a doubling of
short-term C isotope discrimination (Table II). Jenkins et al. (1989)
came to a similar conclusion with respect to CA activity in the
bundle-sheath cytosol. Their model predicted that even a 1000-fold
increase in CA activity (over the nonenzymatic rate) in the cytosol of
the bundle sheath would not have a major effect on the bsc
CO2 concentration or C4
acid pathway overcycling.
| |
CONCLUSIONS |
Previously, biochemical and modeling studies have shown that an
absence of CA in the bundle sheath of C4 plants
is requisite for the effective functioning of the
C4 photosynthetic pathway (Furbank and Hatch,
1987; Burnell and Hatch, 1988). As discussed above, however, the
modeling study of Jenkins et al. (1989) suggested that the presence of
some CA in the bundle sheath would not be as detrimental to the
efficiency of C4 photosynthesis as previously believed. In the present study perturbation of the
CO2-concentrating mechanism of F. bidentis was attempted by expressing mature tobacco CA in the
mesophyll and bsc cytosol. The biochemical and physiological characteristics of the resulting CA transformants indicated that the
CO2-concentrating mechanism of these plants was
less efficient than that of the controls. However, no obvious
phenotypic differences were detected between the CA transformants,
wild-type plants, and the transformed negative controls in any of the
three generations of plants studied.
The expression of tobacco CA in the bundle sheath of F. bidentis resulted in the bsc of the CA transformants containing 3 to 5 times more CA activity than control bsc, and an increase in C
isotope discrimination was measured in the CA transformants both in the
short term during gas exchange and on leaf dry matter. Moreover, the CA
transformants demonstrated reduced net
CO2-assimilation rates relative to the controls.
In light of the relationship predicted between C isotope discrimination
and bsc leakiness (Farquhar, 1983), these data indicate that increasing
the CA activity in the bundle-sheath cytosol of F. bidentis
resulted in plants that are partially defective in the
C4 CO2-concentrating
mechanism due to increased permeability of the bundle sheath to
inorganic C. O2 sensitivity and
PSII measurements from the CA transformants and wild-type plants also indicated that the availability of
CO2 for fixation by Rubisco in the bsc was
decreased in the transformants, and that at supraoptimal
O2 levels, the effectiveness of the
C4 pathway in the transformants was compromised.
The indication that the bundle sheath of F. bidentis
contains more endogenous CA activity than the bsc of other
C4 plants and the intracellular location of this
enzyme activity are currently under further investigation.
| |
FOOTNOTES |
1
Present address: Division of Biochemistry and
Molecular Biology, School of Life Sciences, Australian National
University, Canberra, ACT 0200, Australia.
*
Corresponding author; e-mail martha.ludwig{at}anu.edu.au; fax
61-2-62490313.
Received December 5, 1997;
accepted March 31, 1998.
| |
ABBREVIATIONS |
Abbreviations:
A, CO2 assimilation.
bsc, bundle-sheath cell(s).
CA, carbonic anhydrase.
CaMV, cauliflower mosaic
virus.
Chl, chlorophyll.
ME, malic enzyme.
PSII, quantum
yield of PSII.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the expertise and assistance of Julie
Chitty with the F. bidentis transformation system. We also thank Anthony Millgate and Karin Harrison for their valuable technical support.
| |
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Abstract
Introduction
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