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Plant Physiol. (1998) 117: 733-744
Evolution of C4 Photosynthesis in
Flaveria Species1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C4 photosynthesis, provides CO2 to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C3, C3-C4 intermediate, C4-like, C4) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C4 isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C3 Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C3 photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C4 cycle activity. The 62-kD isoform, which is the predominant highly active form in the C4 species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C4 metabolism, and a 62-kD form that is necessary for the complete functioning of C4 photosynthesis.
Most land plants use the C3 pathway for
carbon fixation, in which each photosynthetic cell uses Rubisco to fix
CO2 directly into C-3 compounds. In
C4 plants, fully differentiated mesophyll and
bundle-sheath cells cooperate to fix CO2 by the
C4 pathway (Edwards and Walker, 1983 The genus Flaveria contains not only
C3 and C4 species, but also
a number of C3-C4
intermediates that have different capacities to reduce photorespiration
by the above mechanisms (Ku et al., 1991 The species used in the study were Flaveria pringlei,
Flaveria robusta, and Flaveria cronquistii
(C3); Flaveria sonorensis, Flaveria oppositifolia, Flaveria angustifolia,
Flaveria linearis, Flaveria floridana, and
Flaveria ramosissima
(C3-C4); Flaveria brownii and Flaveria vaginata
(C4-like); and Flaveria bidentis and
Flaveria trinervia (C4). The species
were propagated vegetatively from shoot cuttings or germinated from
seeds and grown in a compost:sand:perlite mixture (2:1:1, v/v). The
plants were grown in a greenhouse at a 25/18°C day/night thermoperiod
and a 13- to 16-h photoperiod. Maximum illumination on a clear day
during the summer months provided a PPFD of 1750 µmol
m Extraction and Assay of NADP-ME
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Hatch,
1987). In these plants, atmospheric CO2 is first
incorporated into C-4 acids in the mesophyll cells, which are then
transported to bundle-sheath cells, where they are decarboxylated and
the released CO2 is incorporated into organic phosphate by the C3 cycle. The
C4 system is more efficient under some
environmental conditions due to CO2 being
concentrated in bundle-sheath cells that suppresses the oxygenase
activity of Rubisco and, thus, photorespiration.
C3-C4 intermediate species are thought to represent a stage in the evolutionary transition from
the C3 photosynthetic mechanism to
C4 photosynthesis (Monson et al., 1984;
Raws-thorne, 1992). In
C3-C4 species, two
mechanisms are proposed to account for the low apparent
photorespiration (Monson et al., 1984; Rawsthorne, 1992). In all
intermediates, Rubisco is found in both mesophyll and bundle-sheath
cells. The mesophyll cells function as in C3
plants in the fixation of atmospheric CO2 by RuBP
carboxylase via the C3 pathway, and in the RuBP
oxygenase reaction, which is the first step in generating C-2 compounds for the photosynthetic oxidation cycle. In one mechanism of reducing photorespiration that may be common to all intermediates,
photorespiratory metabolites generated as a consequence of the RuBP
oxygenase reaction in mesophyll cells, glycolate and Gly, are
transported to bundle-sheath cells, metabolized through mitochondrial
Gly decarboxylase, and the CO2 released is
refixed by Rubisco in bundle-sheath chloroplasts. Through this means,
reduced photorespiratory CO2 evolution occurs without the operation of a C4 cycle. In some
intermediates, the operation of a limited C4
cycle between the mesophyll and bundle-sheath cells contributes to the
further reduction of photorespiration. These plants exhibit elevated
activities and partial cellular compartmentation of key enzymes of
C4 photosynthesis.
). The
C4 Flaveria spp. have been classified as
the NADP-ME subtype, since this is the major enzyme for decarboxylation
of C4 acids (Ku et al., 1983). Full-length cDNA
clones encoding the enzyme have been isolated from the
C4 species Flaveria trinervia (Borsch
and Westhoff, 1990) and the C3 species
Flaveria pringlei (Lipka et al., 1994). Both cDNA clones
encode proteins of 71 kD in size, which contain 7.9-kD putative transit
peptide sequences for chloroplast targeting of the preproteins. The
size of both mature proteins is about 62 kD. In addition, a partial
cDNA clone has been reported for the
C3-C4 intermediate
Flaveria linearis (Rajeevan et al., 1991). Lipka et al.
(1994) concluded that the gene encoding the C4
NADP-ME isoform descended from a common ancestral gene already present
in C3 species. More recently, Marshall et al.
(1996) isolated three genomic clones of NADP-ME from the
C4 species Flaveria bidentis and
concluded from Southern-blot analysis and sequence comparison with
NADP-ME cDNA clones from other plants that Flaveria spp.
contain three and possibly four NADP-ME genes. They proposed that the
NADP-ME gene family is more complex than previously thought; the two
genomic clones characterized encode two highly similar forms of the
enzyme, one being expressed in C4 photosynthetic
tissue (Me1), whereas the other (Me2) appears to
be constitutively expressed. Genomic Southern blotting with gene-specific probes showed that both Me1 and Me2
are found in C3 and C4
Flaveria spp., and it was suggested that the genes may have
arisen by gene duplication in a common ancestral species. The sizes of
proteins encoded by these two genes have not been determined (W.C.
Taylor, personal communication). Since some evidence exists for a
multigene family for NADP-ME in Flaveria spp., it is
important to evaluate the presence of different isoforms that are
produced in different photosynthetic types of Flaveria at the protein level, and their tissue- and cell-specific localization. In
the present study we detected three isoforms of the enzyme in various
Flaveria spp. One of them, a 72-kD monomer, is found to be
constitutively expressed in photosynthetic and nonphotosynthetic tissues of the different photosynthetic types examined, whereas two
other isoforms, 64- and 62-kD monomers, are only abundant in
photosynthetic tissue having partial or complete
C4 photosynthesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2 s1.
Supplemental light from metal-halide lamps provided a PPFD of 350 µmol m2 s1 on cloudy
days. Plants were fertilized twice per week with Peter's fertilizer
supplemented with micronutrients. The third and fourth pairs of leaves
from the apex were used for protein preparations.
1 fresh weight) and grinding was continued
until total maceration. Crude extracts were filtered through a layer of
Miracloth (Calbiochem) and centrifuged at 15,000g for 5 min.
The supernatant fluid was immediately desalted through a Sephadex G-25
column pre-equilibrated with the extraction medium without PVP. The
desalted extracts were used for enzyme assay. NADP-ME was assayed
spectrophotometrically at 30°C in a mixture containing 50 mM Tris-HCl, pH 8.0, 2.5 mM DTT, 20 mM MgCl2, 0.4 mM NADP,
and 25 to 50 µL of enzyme extract. The reaction was initiated by
adding 5 mM malate (pH 7.8), and the increase in
A340 was recorded.
Suc Density Gradient Centrifugation
For experiments using Suc density centrifugation, leaf protein was extracted from F. cronquistii (9 g), F. brownii (5 g), F. ramosissima, and F. trinervia (both 2 g) as described above. Clarified extracts were obtained by centrifugation at 15,000g for 1 h. The supernatant fraction was brought to 80% saturation with ammonium sulfate at 4°C, stirred for 1 h, and recentrifuged for 15 min. The pellet was resuspended in 2 mL of extraction buffer without PVP and centrifuged at 15,000g for 10 min. The supernatant fluid was diluted with an equal volume of extraction buffer (minus PVP), and 2 mL was loaded onto a 35-mL 10 to 30% (w/v) linear Suc density gradient. The Suc gradient was prepared from 10 and 30% (w/v) stock solutions containing 25 mM Tris-HCl, pH 8.0, 5 mM DTT, 2.5 mM MgCl2, and 0.2 mM EDTA. The gradients were centrifuged at 110,000g for 40 h, and after centrifugation 1-mL fractions were collected and assayed for NADP-ME activity. Peak fractions were pooled for kinetic studies. Suc concentrations were determined using a refractometer.IEF and Activity Staining
For IEF analysis of NADP-ME, leaf protein samples were prepared by grinding the tissue in 100 mM Tris-HCl, pH 7.0, 1 mM EDTA, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, and 2 mM PMSF. Nondenaturing IEF was performed using a 5% (w/v) acrylamide gel with a pH range from 5.0 to 7.0 (samples were loaded on the surface of the gel using small pieces of filter paper). The gels were run for 3 h at 6°C (constant voltage of 0.6 kV) in a LKB 2117 Multiphor system. The pH gradient on the gels was determined by employing a surface pH electrode. NADP-ME on the gels was detected by incubating in a solution containing 50 mM Tris-HCl, pH 7.5, 10 mM L-malate, 10 mg of MgCl2, 0.5 mM NADP, 0.1 mg mL1 nitroblue tetrazolium, and 5 µg
mL1 phenazine methosulfate at room temperature.
SDS-PAGE and Immunoblotting
For western immunoblot studies, total protein from the different tissues was extracted using a phenol extraction procedure according to the work of van Etten et al. (1979). The extraction buffer contained 0.7 M Suc, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% (v/v) 2-mercaptoethanol, 10% (w/v) insoluble PVP, 2 mM PMSF, and 10 µM leupeptin. After total maceration in extraction buffer, an equal volume of water-saturated phenol was added and mixed. Protein that partitioned to the phenol phase was separated from the aqueous phase by centrifugation and precipitated by methanol. Protein was dissolved in 0.25 M Tris-HCl, pH 7.5, 2% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol and boiled for 2 min prior to being loaded onto the gel.. Anti-maize 62-kD NADP-ME IgG (diluted 1:100),
affinity-purified according to the method of Plaxton (1989), was used
for detection (Maurino et al., 1996, 1997). Bound antibodies were
visualized by linking to alkaline phosphatase-conjugated goat
anti-rabbit IgG according to the manufacturer's instructions
(Promega). The molecular masses of the polypeptides were estimated from
a plot of the log of molecular mass of the prestained marker standards
versus migration distance (a linear relationship). The markers and the
samples were run on the same gel.
In Situ Immunolocalization
Transmission Electron Microscopy
Samples for microscopy were fixed for 12 to 24 h at 4°C in 2% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 50 mM Pipes buffer, pH 7.2. The samples were dehydrated with a graded ethanol series and embedded in London Resin White acrylic resin. Thin sections on uncoated nickel grids were incubated for 1 h in TBST (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1% [v/v] Tween 20 [v/v] plus 1% [w/v] BSA) to block nonspecific protein binding on the sections. The sections were then incubated for 16 h with either preimmune serum (without dilution) or affinity purified anti-maize leaf 62-kD NADP-ME IgG (1:10 dilution). After extensive washing with TBST/BSA, the sections were incubated for 1 h with protein A-gold (15 nm) (Amersham) diluted 1:100 with TBST/BSA. The sections were washed with TBST/BSA, TBST, and distilled water prior to poststaining with a 1:4 mixture of 1% (w/v) potassium permanganate and 2% (w/v) aqueous uranyl acetate.Light Microscopy
Sections (1 µm thick) from the same samples prepared for transmission electron microscopy were dried onto gelatin-coated slides and blocked for 1 h with TBST/BSA. They were then incubated 16 h with the purified antibody or preimmune serum with TBST/BSA. The slides were washed and then treated for 1 h with protein A-gold. The sections were subsequently exposed to a silver enhancement reagent according to the manufacturer's directions (Amersham), stained with 1% (w/v) Safranin O, and photographed using an Aristoplan microscope (Leitz).Assays of Protein and Chlorophyll
Protein concentration was determined by the method of Sedmak and Grossberg (1977) using BSA as a standard. Chlorophyll was determined
after extraction in 96% (v/v) ethanol according to the method of
Wintermans and De Mots (1965).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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NADP-ME Activity in Leaves of Flaveria spp.
Leaf extracts from Flaveria spp. representing the different photosynthetic types were assayed for NADP-ME activity. There was a progressive increase in activity from C3 to C3-C4, C4-like, and C4 with average values of 131, 188, 870, and 1224 µmol mg1 chlorophyll h1,
respectively (data for individual species not shown). However, there
was a range of activities among the intermediate species from
C3-like values to higher values (ranging from
85-359 µmol mg1 chlorophyll
h1). Among the
C3-C4 intermediates having
the lowest activity, F. sonorensis did not have a functional
C4 cycle, and F. angustifolia and
F. linearis had very low C4 cycle
activity; the highest NADP-ME activity occurred in F. ramosissima, which had the highest degree of function of a limited
C4 cycle in this photosynthetic group (Monson et
al., 1986; Moore et al., 1987; Ku et al., 1991). The data, combined
with that of Ku et al. (1991), show a clear relationship between the
level of NADP-ME activity and other characters that define the degree
of C4 photosynthesis in the different
Flaveria spp.
Separation of NADP-ME by Suc Density Gradient Centrifugation
A representative species of each photosynthetic type (C3, F. cronquistii; C3-C4, F. ramosissima; C4-like, F. brownii; and C4, F. trinervia) was used for separation of leaf-soluble protein on Suc density gradients and subsequent fractionation and assay of NADP-ME activity (Fig. 1). After centrifugation, proteins with higher molecular masses resided at higher Suc densities. Although resolution of proteins with small differences in mass were limited by this technique, there was evidence for multiple forms of NADP-ME. In C3 F. cronquistii, two peaks of NADP-ME activity appeared, with the higher mass form (peak fraction 19 at 19.5% Suc) being predominant compared with the lower- mass form (peak fraction 13 at about 16.5% Suc), whereas in C4 F. trinervia there was a major lower-mass form (peak fraction 12 at about 16.5% Suc) and a small shoulder of activity appearing at a higher density. In the C3-C4 F. ramosissima, there was evidence for two dominant forms (around 19.5 and 16.5% Suc), whereas in the C4-like species F. brownii, only one major peak was apparent, with maximum activity occurring in fraction 15 (17.5% Suc). These results suggest that isoforms of NADP-ME with different molecular masses likely exist among the Flaveria spp., and that the relative abundance of the isoforms may vary between the different photosynthetic types.
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Immunoblot Analysis of NADP-ME in Different Flaveria spp.
Total proteins from leaves of 13 species of Flaveria were extracted by a phenol procedure in the presence of two proteinase inhibitors and a sulfhydryl reagent to minimize proteolytic cleavage during preparation. These protein extracts were electrophoretically separated in SDS gels, transferred onto nitrocelluose membranes, and probed with affinity-purified antibody prepared against NADP-ME from maize leaves. The maize NADP-ME purified from green leaves is specific for C4 photosynthesis and has a molecular mass of 62 kD (Maurino et al., 1996). This antibody reacts against the photosynthetic isoform of
NADP-ME (62-kD monomeric molecular mass) and a nonphotosynthetic
isoform (72-kD monomeric molecular mass) present in green and etiolated
maize leaves, in maize roots, and in C3 plants
such as wheat (Maurino et al., 1996, 1997).
Isoform Pattern of NADP-ME as Resolved by IEF
Immunolocalization Studies
Isoforms of NADP-ME in Flaveria spp.
C4 and C3 Species
C3-C4 Species
; Ku et al., 1983; Edwards and Ku, 1987).
In the C3-C4 intermediate
Flaveria spp., the antibody reacted with one to three
different molecular mass monomers of 72, 64, and 62 kD, which was
species dependent, with all species having a 72-kD isoform. In the case
of F. sonorensis, the only reactive band was at 72 kD (Fig.
2, lane 6). This plant has low apparent photorespiration without
C4 photosynthesis, and Rubisco and the
C3 pathway are considered to function in
mesophyll cells in the same way as in C3 plants,
with refixation of photorespired CO2 by Rubisco
in bundle-sheath cells (Ku et al., 1991). The other five
C3-C4 intermediate plants
that exhibit a varying capacity for C4
photosynthesis (Ku et al., 1991) have in addition to the 72-kD form of
the enzyme, a 62- and/or a 64-kD reactive form. F. angustifolia (Fig. 2, lane 8) had the 72- and 62-kD forms. All
three forms, 72, 64, and 62 kD, are apparent in F. oppositifolia (Fig. 2, lane 7), F. floridana (Fig. 2,
lane 10), and F. linearis (Fig. 2, lane 11). F. ramosissima expressed the 72- and the 64-kD forms, with a low
level of the 62-kD form (Fig. 2, lane 9).
View larger version (30K):
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Figure 2.
Immunoblot analysis of leaf total proteins
extracted from different Flaveria spp. Leaf proteins of
Flaveria spp. (60 µg) and maize (10 µg) were
separated by SDS-PAGE, transferred onto nitrocellulose membranes, and
probed with purified anti-maize 62-kD NADP-ME IgG. The lines indicate
the position of the 72-, 64-, and 62-kD immunoreactive bands. The
species used were C4: maize (lane 1); F. trinervia (lane 2); F. bidentis (lane 3).
C4-like: F. brownii (lane 4); F. vaginata (lane 5). C3-C4: F. sonorensis (lane 6); F. oppositifolia (lane 7);
F. angustifolia (lane 8); F. ramosissima
(lane 9); F. floridana (lane 10); F. linearis (lane 11). C3: F. cronquistii (lane 12); F. pringlei (lane 13);
F. robusta (lane 14).
; Moore et al., 1989). In the C4 species
F. trinervia (Fig. 2, lane 2) and F. bidentis (Fig. 2, lane 3), the most reactive band was the 62-kD monomer. Nevertheless, a faint 72-kD form was still present and the 64-kD form
was expressed at very low levels. The isoform pattern of NADP-ME in
C4 Flaveria spp. is similar to that of
maize, which has a predominant 62-kD form and little or no 64-kD
form.
).
However, the results show a preferential expression of the three
isoforms of NADP-ME with different photosynthetic mechanisms in
Flaveria spp.
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Figure 3.
Relative abundance of the three monomeric isoforms
of NADP-ME in the different photosynthetic types of
Flaveria spp.. The western blots from the results in
Figure 2 were scanned, the areas of the peaks corresponding to the
monomeric forms were determined, the average of each form within each
photosynthetic type was calculated, and the relative abundance was
determined. Immunoblots were repeated three times with the same phenol
extract for all species; duplicate extracts from separate leaves with a
species representing each photosynthetic type gave similar results. The
results are presented for each form as a percentage of the maximum,
with the photosynthetic group having the maximum amount of that form
taken as 100%. Very low levels of the 62-kD (black columns) and 64-kD
(striped columns) forms were detected in scans of the C3
species, although they are not apparent in the immunoblots in Figure 2
(lanes 12-14). The white columns represent 72-kD forms.
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Figure 4.
Activity staining of leaf NADP-ME from F. pringlei (C3), F. floridana
(C3-C4 intermediate), and F. trinervia (C4) on native IEF gels. The pH gradient
used for IEF was from 5.0 to 7.0. The calculated native pI of the
reactive bands are between 5.3 and 5.8. Leaf protein extracts were made
from F. pringlei (lane 1), F. floridana (lane 2), and F. trinervia (lane 3).
The amount of protein loaded was equivalent to 1 milliunit of NADP-ME
activity.
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Figure 5.
Light microscopy of in situ immunolocalization of
NADP-ME in leaves of F. bidentis (C4, upper
panel), F. robusta (C3, lower left panel),
and F. ramosissima (C3-C4, lower
right panel).
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Figure 6.
Electron microscopy of in situ immunolocalization
of NADP-ME in leaves of Flaveria spp. A to C, F. bidentis (C4); D, F. robusta (C3); E and F, F. ramosissima
(C3-C4). bsc, Bundle-sheath cell; mc, mesophyll
cell; cc, companion cell.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Maurino
et al., 1997). Maize leaves also have a native form of higher mass that
is constitutively expressed in roots, etiolated leaves, and at low
levels in green leaves, and this form corresponds to the higher-mass
form in wheat (Maurino et al., 1997). The higher-mass form of the
enzyme in C3 species of Flaveria
appears to have a higher Km value for NADP than the lower-mass form in C4 species of
Flaveria, which is consistent with reports
that the enzyme from leaves of C3 and
C4 species has different kinetic properties (see
Edwards and Andreo, 1992).
) and to the larger molecular mass, constitutive
72-kD monomer of the enzyme that occurs in maize roots, etiolated
leaves, and at low levels in green leaves (Maurino et al., 1997).
Previously, Cameron et al. (1989) reported no immunoreactivity of
NADP-ME in leaf extracts of C3 F. pringlei
in western blots using antibodies against the maize enzyme, which may
be due to differences in the epitopes that the antibodies prepared with
the maize 62-kD protein can recognize (also see Maurino et al., 1997).
; Maurino et al., 1997). Thus, in
leaves, stems, and roots of C3 species of
Flaveria and in stems and roots of
C4 Flaveria spp., the apparent predominant
form of the enzyme may consist of a homotetramer of 72-kD subunits,
whereas in leaves of C4 Flaveria spp. the
apparent predominant form of the enzyme may be a homotetramer of 62-kD
polypeptides. These results are consistent with the native form of the
enzyme existing as one dominant band of activity in nondenaturing IEF
gels in C3 and C4 species
of Flaveria, but with different pI (Fig. 4). So far in
Flaveria spp., NADP-ME genes have been cloned
from the C3 F. pringlei (Lipka et al.,
1994) and the C4 F. trinervia (Borsch and Westhoff, 1990), and both encode a subunit with deduced molecular mass of 62 kD. The genomic clones of NADP-ME isolated from another C4 species, F. bidentis, remain to be
characterized in terms of the sizes of their protein products (Marshall
et al., 1996).
), the 72-kD form is
predominant, which is similar to the situation in
C3 species. In other intermediates, where there
is evidence for a limited degree of functioning of C4 photosynthesis, such as F. ramosissima,
F. linearis, and F. floridana (Monson et al., 1986;
Moore et al., 1987; Ku et al., 1991), multiple forms of NADP-ME are
present. Previously, Cameron and Basset (1988) suggested from western
analysis that there is only one form of NADP-ME in leaves of
intermediates (F. floridana, F. oppositifolia, and F. linearis), but these results were inconclusive due to the poor
reactivity of their antibody.
C4-Like Species
In leaves of the C4-like species F. brownii, there was one major band of NADP-ME activity on a Suc density gradient (Fig. 1C); however, two major bands of 62 and 64 kD were revealed on western blots of SDS gels (Fig. 2, lane 4). Thus, this C4-like species has a significant amount of the 64-kD form, which occurs to a lesser extent in intermediates (Fig. 3), and a 62-kD form, which is the major form in C4 species. The C4-like species F. vaginata also has two major forms on SDS gels; however, these are slightly smaller than the 64- and 62-kD forms in F. brownii.Localization of NADP-ME in Flaveria spp. Leaves
C4 and C3 Species
Immunolocalization studies of leaves of C4 species of Flaveria show that the major labeling occurs in bundle-sheath chloroplasts, with minor labeling in mesophyll chloroplasts (Figs. 5 and 6). This is consistent with the C4 form of the enzyme having an essential function in the bundle-sheath cells of certain C4 plants in donating CO2 to RuBP carboxylase. In studies with isolated mesophyll and bundle-sheath protoplasts of C4 F. trinervia, it was shown that high NADP-ME activity occurs in bundle-sheath protoplasts (40-fold higher activity than in mesophyll protoplasts), with compartmentation of the enzyme in the chloroplast (Moore et al., 1984); recently it was shown that isolated bundle-sheath chloroplasts of F. bidentis have high activity of NADP-ME (Meister et al., 1996). In
maize, bundle-sheath chloroplasts have high levels of the 62-kD form, which provides direct evidence for its role in C4
photosynthesis, whereas the mesophyll chloroplasts have only low levels
of the constitutive 72-kD form (Maurino et al., 1997). Thus, by
analogy, the low level of labeling in the mesophyll chloroplasts of
F. trinervia may be attributed to the constitutive 72-kD
form.
).
C3-C4 Intermediate Species
Evidence that intermediates have incomplete development of C4 photosynthesis at the biochemical level was previously shown by immunocytochemical studies with several C3-C4 Flaveria spp. (F. linearis, F. floridana, and F. chloraefolia). Neither Rubisco nor PEPCase was specifically located in one cell type, as is the case in C4 species, but rather they each occurred in both mesophyll and bundle-sheath cells (Reed and Chollet, 1985). In the present study,
major immunolabeling of NADP-ME was found in bundle-sheath chloroplasts, whereas significant labeling also occurred in mesophyll chloroplasts of the C3-C4
intermediate F. ramosissima (Figs. 5 and 6), a species that
has substantial C4 cycle function (see Ku et al.,
1991). The distribution of the three forms identified on western blots
between the two cell chloroplast types is not known. It was previously
reported (Moore et al., 1988) that within the leaf of this
C3-C4 plant there is a
gradation of decreasing PEPCase activity along with increasing NADP-ME
and Gly decarboxylase activities from the peripheral mesophyll cells to
the bundle-sheath cells. In this way, apparent photorespiration may be
reduced by two mechanisms in this species, one through concentrating
CO2 by C4 acid
decarboxylation in the bundle-sheath and the other through refixation
of the photorespired CO2 in the bundle-sheath cells. Although the compartmentation of the different forms of NADP-ME
in the leaves of C3-C4
intermediate and C4-like Flaveria spp.
is uncertain, if the 62- and 64-kD forms are associated with the
C4 cycle they may be enriched in the
bundle-sheath chloroplasts.
Isoforms and Gene Family of NADP-ME in Flaveria spp.
Marshall et al. (1996) showed that the C3
F. pringlei and the C4 F. bidentis and
F. trinervia have at least three and perhaps four NADP-ME
genes. In examining two of these genes from F. bidentis, Me1 and Me2, in detail, they concluded that these
genes are very similar in sequence, that both are present in
C3 and C4 species of
Flaveria, and that they both encode
putative transit peptide sequences for targeting their preproteins into
chloroplasts. They suggested that these are paralogous genes arising by
gene duplication from a common ancestral species. Also, from
examination of Me1 and Me2 mRNA levels there was
evidence that Me1 encodes the C4 form
of the enzyme in leaves (leaf-specific, light-dependent expression in
C4 plants only) and that Me2 is
constitutively expressed (lack of organ specificity in expression, low
level of expression). The 5
and 3 flanking regions of the
Me1 gene are important for bundle-sheath specificity and
high-level expression in leaves, respectively (Marshall et al., 1997).
Thus, at least two chloroplastic forms of NADP-ME might be expected to
occur in Flaveria spp. It is reasonable to suggest from the
results of the present study that Me1 encodes the 62-kD form
that is targeted to bundle-sheath chloroplasts in
C4 species and that Me2 encodes the
constitutive 72-kD form that is targeted to leaf plastids and stem and
root tissues of both C3 and
C4 species. In addition, the present study identified a third form of the enzyme in Flaveria spp., a
64-kD form that is most abundant in C4-like and
certain C3-C4 intermediate species and occurs in green leaf tissue but not in roots and stems of the intermediate species examined. Thus, this may be the product of
a third NADP-ME gene, which is more actively expressed during evolutionary transition from C3 to
C4 photosynthesis. Sequencing of these three
different monomeric forms will be required to match proteins to the
NADP-ME genes and cDNAs that have been isolated.
). These include changes in cell-specific
expression (e.g. carbonic anhydrase), gene duplication coupled with
acquisition of strong promoters for high-level expression (e.g.
PEPCase), and acquisition of strong promoters for high-level expression of chloroplast-specific isoforms in a cell-specific manner (e.g. pyruvate, Pi dikinase). In this study we have demonstrated the presence
of three chloroplastic NADP-ME isoforms, presumably encoded by three
different isogenes, in leaves of all types of photosynthetic Flaveria spp. (C3,
C3-C4,
C4-like, and C4), but
differing in relative abundance (Figs. 3, 5, and 6). These results
suggest that a differential expression of the existing NADP-ME genes
encoding the chloroplastic forms in a tissue- and cell-specific manner
was involved in the evolution of C4
photosynthesis in the genus Flaveria. The genes encoding the
62- and 64-kD forms must have been altered for increased expression
during the evolution of C4 photosynthesis, and in
the case of the 62-kD form, it is also clear that these alterations must have also included an element for bundle-sheath-specific expression.
| |
FOOTNOTES |
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Received December 29, 1997;
accepted April 6, 1998.
| |
ABBREVIATIONS |
|---|
Abbreviations: NADP-ME, NADP-malic enzyme. PEPCase, PEP carboxylase. RuBP, ribulose 1,5-bisphosphate.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Borsch D, Westhoff P (1990) Primary structure of NADP-dependent malic enzyme in the dicotyledonous Flaveria trinervia. FEBS Lett 273: 111-115 [CrossRef][Medline]
Burnette WN (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195-203 [CrossRef][Web of Science][Medline]
Cameron RG,
Bassett CL
(1988)
Inheritance of C4 enzymes associated with carbon fixation in Flaveria species.
Plant Physiol
88:
532-536
Cameron RG,
Bassett CL,
Bouton JH,
Brown RH
(1989)
Transfer of C4 photosynthetic characters through hybridization of Flaveria species.
Plant Physiol
90:
1538-1545
Casati P,
Spampinato CP,
Andreo CS
(1997)
Characteristics and physiological function of NADP-malic enzyme from wheat.
Plant Cell Physiol
38:
928-934
Edwards GE, Andreo CS (1992) NADP-malic enzyme from plants. Phytochemistry 31: 1845-1857 [CrossRef][Web of Science][Medline]
Edwards GE, Ku MSB (1987) The biochemistry of C3-C4 intermediates. In MD Hatch, K Boardman, eds, The Biochemistry of Plants, Vol 14. Academic Press, New York, pp 275-325
Edwards GE, Walker DA (1983) C3, C4: Mechanisms, and Cellular and Environmental Regulation of Photosynthesis. Blackwell Scientific Publishers, Oxford, UK
Hatch MD (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895: 81-106
Ku MSB, Kano-Murakami Y, Matsuoka M (1996) Evolution and expression of C4 photosynthesis genes. Plant Physiol 111: 949-957 [CrossRef][Web of Science][Medline]
Ku MSB,
Monson RK,
Littlejohn RO,
Nakamoto H,
Fisher DB,
Edwards GE
(1983)
Photosynthetic characteristics of C3-C4 intermediate Flaveria species. I. Leaf anatomy, photosynthetic responses to O2 and CO2, and activities of key enzymes in the C3 and C4 pathways.
Plant Physiol
71:
944-948
Ku MSB,
Wu J,
Dai Z,
Scott RA,
Chu C,
Edwards GE
(1991)
Photosynthetic and photorespiratory characteristics of Flaveria species.
Plant Physiol
96:
518-528
Lipka B, Steinmuller K, Rosche E, Borsch D, Westhoff P (1994) The C3 plant Flaveria pringlei contains a plastidic NADP-malic enzyme which is orthologous to the C4 isoform of the C4 plant F. trinervia. Plant Mol Biol 26: 1775-1783 [CrossRef][Web of Science][Medline]
Marshall JS,
Stubbs JD,
Chitty JA,
Surin B,
Taylor WC
(1997)
Expression of the C4 Me1 gene from Flaveria bidentis requires an interaction between 5 and 3 sequences.
Plant Cell
9:
1515-1525
[Abstract]
Marshall JS, Stubbs JD, Taylor WC (1996) Two genes encode highly similar chloroplastic NADP-malic enzymes in Flaveria. Plant Physiol 111: 1251-1261 [Abstract]
Maurino VG, Drincovich MF, Andreo CS (1996) NADP-malic enzyme isoforms in maize leaves. Biochem Mol Biol Int 38: 239-250 [Web of Science][Medline]
Maurino VG, Drincovich MF, Casati P, Andreo CS, Edwards GE, Ku MSB, Gupta SK, Franceschi VR (1997) NADP-malic enzyme: immunolocalization in different tissues of the C4 plant maize and the C3 plant wheat. J Exp Bot 48: 799-811
Meister M, Agostino A, Hatch MD (1996) The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.). Planta 199: 262-269
Monson RK, Edwards GE, Ku MSB (1984) C3-C4 intermediate photosynthesis in plants. BioSci 34: 563-574
Monson RK, Moore Bd, Ku MSB, Edwards GE (1986) Co-function of C3 and C4-photosynthetic pathways in C3, C4 and C3-C4 Flaveria species. Planta 168: 493-502 [CrossRef]
Moore Bd, Ku MSB, Edwards GE (1984) Isolation of leaf bundle-sheath protoplasts from C4 dicot species and intracellular location of selected enzymes. Plant Sci Lett 35: 127-138
Moore Bd, Ku MSB, Edwards GE (1987) C4 photosynthesis and light dependent accumulation of inorganic carbon in leaves of C3-C4 and C4 Flaveria species. Aust J Plant Physiol 14: 657-668
Moore Bd, Ku MSB, Edwards GE (1989) Expression of C4-like photosynthesis in several species of Flaveria. Plant Cell Environ 12: 541-549
Moore Bd,
Monson RK,
Ku MSB,
Edwards GE
(1988)
Plant Cell Physiol
29:
999-1006
Plaxton WC (1989) Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil plant endosperm and leaf. Eur J Biochem 181: 443-451 [Web of Science][Medline]
Powell AM (1978) Systematics of Flaveria (Flaveriinae-Asteraceae). Ann Missouri Bot Garden 65: 591-636
Rajeevan MS, Basset CL, Hughes DW (1991) Isolation and characterization of cDNA clones for NADP-malic enzyme from leaves of Flaveria: transcript abundance distinguishes C3, C3-C4 and C4 photosynthetic types. Plant Mol Biol 17: 371-383 [Medline]
Rawsthorne S (1992) C3-C4 intermediate photosynthesis: linking physiology to gene expression. Plant J 2: 267-274 [CrossRef]
Reed JE, Chollet R (1985) Immunofluorescent localization of phophoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase proteins in leaves of C3, C4 and C3-C4 intermediate Flaveria species. Planta 165: 439-445 [CrossRef]
Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brillant blue G250. Anal Biochem 79: 544-552 [CrossRef][Web of Science][Medline]
Van Etten JL,
Freer SN,
McCune BK
(1979)
J Bacteriol
138:
650-652
Wintermans JFGM, De Mots A (1965) Spectrophotometric characteristics of chlorophylls and their pheophytins in ethanol. Biochim Biophys Acta 109: 448-453 [Medline]
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) is shown on the left
y-axes and the percent Suc in the gradient (
) is
shown on the right y-axes.