Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4 System of Hydrilla verticillata1
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Photosynthetic and Other Phosphoenolpyruvate
Carboxylase Isoforms in the Single-Cell, Facultative C4
System of Hydrilla verticillata1
Srinath K. Rao, Noël C. Magnin2, Julia B. Reiskind, and George Bowes*
Department of Botany, 220 Bartram Hall, P.O. Box 118526, University of Florida, Gainesville, Florida
32611–8526
The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C4 plant. It typically exhibits C3 photosynthetic
characteristics, but exposure to low [CO2] induces a C4 system in which the C4 and Calvin cycles co-exist in the same cell
and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs
encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99%
identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4,
was exclusively expressed in leaves during C4 induction. This and enzyme kinetic data were consistent with it being the C4
photosynthesis isoform. However, the C4 signature serine of terrestrial plant C4 isoforms was absent in this and the other
H. verticillata sequences. Instead, alanine, typical of C3 sequences, was present. Western analyses of C3 and C4 leaf extracts
after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses,
the sequences grouped with C3, non-graminaceous C4, and Crassulacean acid metabolism PEPCs but not with the
graminaceous C4, and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of
other C4 angiosperms.
Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC Investigations on the origins of the C4 syndrome
4.1.1.31) occurs in eubacteria, cyanobacteria, green indicate that it arose independently in a number of
algae, and all higher plants. In the latter, it is encoded angiosperm taxa and included changes in the genes
by a small multigene family (Lepiniec et al., 1993, controlling anatomical and chloroplastic develop-
1994; Ernst and Westhoff, 1997). A major function of ment and in those orchestrating photosynthetic bio-
the enzyme in higher plants is anapleurotic, provid- chemistry (Hermans and Westhoff, 1992; Kellogg,
ing carbon skeletons for the synthesis of compounds 1999). PEPC has played an important role in these
that serve in processes such as C/N partitioning, studies, and the structure, function, and phylogenetic
guard cell movements, and nitrogen fixation in le- relationships of its sequences have been used to bet-
gumes (Chollet et al., 1996). In C4 and Crassulacean ter understand the evolution of C4 and CAM photo-
acid metabolism (CAM) photosynthesis, alternate synthetic systems (Lepiniec et al., 1994).
forms of PEPC catalyze the initial carboxylation The aquatic monocot Hydrilla verticillata (L.f.) Royle
step in a C4 acid cycle that functions as a CO2 con- is the best documented case of an inducible C4 pho-
centrating mechanism. In terrestrial C4 plants, PEPC tosynthetic system that concentrates CO2 in the chlo-
and Rubisco fixation events are separated between roplasts without enzymatic compartmentation in me-
mesophyll and bundle sheath cells, and PEPC expres- sophyll and bundle sheath cells, i.e. it lacks Kranz
sion is in the cytosol of the former (Matsuoka and anatomy. When [CO2] is abundant, H. verticillata ex-
Sanada, 1991). In CAM plants, the fixation events are hibits C3 characteristics, but a C4 photosynthetic sys-
separated temporally with the CAM photosynthetic tem is induced by exposure to low [CO2], both in
PEPC expressed in the cytosol of chloroplastic cells nature and in the laboratory. Thus, it is best de-
(Cushman and Bohnert, 1999). scribed as a facultative NADP-ME C4 species (Bowes
et al., 2002). The induction has been demonstrated by
1
This work was supported by the National Science Foundation gas exchange and biochemistry, 14C pulse-chase
(grant no. IBN–9604518) and by the U.S. Department of Agricul- studies, enzyme localization, and measurements of
ture National Research Initiatives Competitive Grants Photosyn- internal [CO2] (Bowes and Salvucci, 1989; Magnin et
thesis and Respiration Program (grant no. 93–37306 –9386). al., 1997; Reiskind et al., 1997). A unique trait of this
2
Present address: Université Victor Segalen Bordeaux 2, Centre system is that the C4 and Calvin cycles exist together
de Bio-Informatique, 146 Rue Léo Saignat, 33076, Bordeaux,
within the same cell, and the site of CO2 concentrat-
France.
* Corresponding author; e-mail gbowes@botany.ufl.edu; fax ing is the leaf mesophyll chloroplasts (Reiskind et al.,
352–392–3993. 1997). Global climate change scenarios predicting
Article, publication date, and citation information can be found drought and high temperatures have heightened in-
at www.plantphysiol.org/cgi/doi/10.1104/pp.008045. terest in the regulation and expression of the suite of
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© 2002 American Society of Plant Biologists
Copyright © 2002 American Society of Plant Biologists. All rights reserved.Phosphoenolpyruvate Carboxylase Isoforms in Hydrilla verticillata
enzymes involved in C4 photosynthesis. A goal of summarized in Table I. The 5⬘ region in all of the
such research is to introduce C4 cycle components into isoforms had two ATG triplets that are candidates for
C3 crop species with the hope that the transformants, translation initiation; the two different coding se-
similar to C4 and CAM plants, would have improved quence lengths that would occur with each of the
performance under adverse conditions (Matsuoka ATG triplets are also shown. These data indicate that
and Sanada, 1991; Ku et al., 1999; Mann, 1999). In this the encoded proteins were very similar in terms of Mr
context, H. verticillata provides a higher plant exam- and pI.
ple, albeit an aquatic one, of how the C4 and Calvin A comparison of the nucleotides (nt) from the 5⬘-
cycle components might co-exist in the same cell and and 3⬘-untranslated regions (UTR) of the three H.
still function in series to concentrate CO2. verticillata PEPC cDNA sequences indicates that
As part of a molecular approach to understand Hvpepc3 and Hvpepc5 were very similar but not iden-
how the C4 system in H. verticillata is induced and tical and that they differed from Hvpepc4. The 5⬘-UTR
regulated, we have focused attention on the PEPC of Hvpepc5 showed 1 bp deletion and one substitu-
isoforms that we have found in this plant. We present tion compared with Hvpepc3, whereas there were 2
evidence that one is induced and operates in C4 leaf bp substitutions in the 3⬘-UTR and 4 bp substitutions
photosynthesis. Multiple isoforms are commonly re- in the coding region. The Hvpepc5 sequence down-
ported for PEPC gene families (Ernst and Westhoff, stream of the stop codon (TAA) was 116 bp shorter
1997). However, this is the first report of three full- than that of Hvpepc3. All three sequences contained a
length PEPC cDNAs isolated from a plant that is single polyadenylation signal motif.
normally C3, but has evolved an inducible C4 system A comparative multiple alignment of the deduced
to combat the adverse environmental conditions of amino acid sequences of the three H. verticillata
low [CO2] and high [O2], temperature, and irradiance PEPCs with those of two other monocots and one
that occur during summer days (Bowes and Salvucci, eudicot representing C3, C4, and CAM isoform se-
1989). The phylogenetic relationships of H. verticillata quences is shown in Figure 1. The monocot maize
PEPC isoforms with those of members of other spe- contains both C3 and C4 PEPCs, whereas the monocot
cies possessing C3, C4, and CAM isoform types are Vanilla planifolia has a CAM isoform. The C4 PEPC
also shown. from the C4 eudicot F. trinervia was also included in
the comparison because this sequence bears a phylo-
RESULTS genetic resemblance to those of H. verticillata. The
conserved regions for both eukaryotic and prokary-
Isolation, Cloning, and Sequencing of Three
otic PEPCs are indicated, as well as the specific cat-
Full-Length cDNAs Encoding H. verticillata PEPC
alytic and regulatory binding locales and two puta-
Hvpepc3 and 4 were culled from 40 C4 leaf-derived tive C4 signature sites. Homology among the H.
RACE clones that screened positively for either the verticillata sequences was high (95%–99%), and they
3F or 4F oligoprobe. Subsequent isolations using C3 showed the closest resemblance to the C3 PEPC from
leaf material yielded only clones of Hvpepc3. A sim- maize (85%). Identity with the CAM PEPC was 83%,
ilar number of root-derived RACE clones tested pos- with the F. trinervia C4 PEPC 81%, and with the maize
itively only to the probe 3F, and from these clones, C4 PEPC 78%. In a comparison with Hvpepc3, Hvpepc5
Hvpepc5 was isolated and sequenced. The salient fea- had three substitutions resulting from the 4 bp
tures of these cDNAs and their encoded PEPCs are changes, whereas Hvpepc4 had 44 substitutions and
Table I. Characteristics of the cDNAs and the predicted amino acid sequences of the three PEPC
isoforms from H. verticillata
Three full-length cDNAs encoding PEPC were sequenced using RACE-PCR techniques. The percent
identities among the nucleotide and amino acid sequences were measured by pair-wise comparison.
The data for Hvpepc4 and 5 are compared with those of Hvpepc3.
Hvpepc3 Hvpepc4 Hvpepc5
cDNA (bp) 3,368 3,197 3,251
Coding sequence 60 –2,972 62–2,968 59 –2,971
99 –2,972 95–2,968 98 –2,971
5⬘-UTR (bp) 1–59 (59) 1– 61 (61) 1–58 (58)
Percent nucleotide identity at 5⬘-UTR (bp) 100 80 98
3⬘-UTR (bp) 2,973–3,368 2,969 –3,197 2,972–3,251
(395) (228) (279)
Percent nucleotide identity at 3⬘-UTR (bp) 100 67 99
Deduced amino acids 970 968 970
Percent amino acid identity 100 95 99
Molecular mass 110,345 109,970 110,344
pI 6.4 6.4 6.4
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Figure 1. Multiple alignment of the deduced amino acid sequences of three H. verticillata PEPCs and those of maize (Zea
mays; C3 and C4), Vanilla planifolia (CAM), and Flaveria trinervia (C4). Only residues that differ among the sequences are
shown. Gaps (-) and identical (.) bp are indicated. Boxed residues indicate the most conserved regions among prokaryotes
and eukaryotes. Putative regulatory and catalytic sites are also shown. 佣, The Ser residue that is common to all plant PEPCs
and that is the target for phosphorylation; F, the unique Hvpepc4 Met residue; 佧, the F. trinervia putative C4 Lys site; E,
the unique Hvpepc5 Val; and 夹, the position of the C4 signature Ser.
two deletions. The three substitutions found in is found in all other PEPCs listed in the database. The
Hvpepc5 were Ser-196 for Cys, Val-777 for Ile, and signature C4 Ser, Ser-774 of F. trinervia (Bläsing et al.,
Arg-891 for Glu. The substitutions in Hvpepc4 oc- 2000), was also present in the C4 PEPC of maize, but
curred mostly in the variable regions; the Met-150 it was notably absent from all of the H. verticillata
appears to be a unique change, replacing Leu, which sequences. Instead, Ala was found at the correspond-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.Phosphoenolpyruvate Carboxylase Isoforms in Hydrilla verticillata
ing position. A putative C4-determinant Lys-347, as recommended (Roche Diagnostics/Roche Applied
described for the F. trinervia C4 PEPC, occurred in Science, Indianapolis). The loading of greater quan-
Hvpepc4 at the same position, whereas the putative tities of total RNA (2 and 5 g) did not change the
C3-marker Arg occurred in the other H. verticillata detection threshold. The probe to the Hvpepc3 iso-
isoforms (Hermans and Westhoff, 1992; Bläsing et al., form was specifically synthesized from its 3⬘-UTR,
2000). It should be noted that Lys-347 is not an ab- however the similarity between these regions of
solute C4 marker, because it also occurs in CAM and Hvpepc3 and Hvpepc5 suggests that the probe could
some C3 sequences and not in the graminaceous C4 not discriminate between these two isoforms. There-
PEPC isoforms. None of the other reported C4- fore, an Hvpepc5 signal in the C3 leaves cannot be
determinant residues described by Hermans and excluded.
Westhoff (1992) were found in the H. verticillata de- The activity of PEPC was followed in the same
duced sequences. samples used for the northern analyses. Figure 3
shows the specific activity over time of PEPC in
desalted extracts from the C3- and C4-induced leaves
Differential Expression of H. verticillata Isoforms and shows the times when RNA was sampled for the
northern analyses. The PEPC activity in the C3 leaves
To compare the specific expression of Hvpepc3 and remained essentially constant and low. In contrast,
Hvpepc4, northern analyses were performed using C3 that of the C4-induced leaves increased in a linear
and C4 leaves of H. verticillata (Fig. 2). The samples fashion, reaching values more than 10-fold greater
were analyzed several times throughout the C4 in- than in the C3 leaves.
duction period, starting at zero time when all the
leaves had C3 photosynthetic characteristics. When
isoform-specific RNA probes were used, Hvpepc4 was Partial Purification of PEPC, Kinetic
expressed exclusively in C4-induced leaves, after 96 Characterization, and Western Analyses
and 264 h into the induction period. This isoform
notably was not expressed in any other samples. In Data for the purification of PEPC from extracts of
contrast, Hvpepc3 was expressed in C3 and C4 leaf C3 and C4 leaves (harvested in the light at 288 h into
samples, except at the 264-h C3 sampling time. The the induction period) and roots, using ammonium
results of consensus probing were similar to those sulfate fractionation and Q-Sepharose FF anion-
using the Hvpepc3 probe. The results represent a 1-g exchange chromatography, are summarized in Table
total RNA loading scheme, which is the maximum II. The PEPC activities were assayed at the optimal
pH of 8.0 with saturating substrate concentrations.
The root extract did not bind to the column but
eluted as a single peak in the buffer wash. However,
the leaf extracts did bind and were eluted with a
linear salt gradient. The elution profiles of each of
these extracts were characterized by a single peak,
but with elution at slightly different salt concentra-
tions. The specific PEPC activities in both the crude
and chromatographed C4 leaf extracts were substan-
tially higher than the corresponding C3 values, and
leaf values were much higher than those of the roots.
The crude activities were similar to those described
previously (Fig. 3). The purification factors were
greater for the leaf extracts than for the root.
Kinetic data for the C3 and C4 leaf peak PEPC
fractions are presented in Figure 4. The activities
were assayed at a cytosolic-like pH of 7.3, where
PEPC kinetic effects are more pronounced. A plot of
activity versus [PEP] produced a hyperbolic curve for
the C3 leaf enzyme that followed Michaelis-Menten
Figure 2. Northern analyses of PEPC isoform expression in H. verti- kinetics (r2 ⫽ 0.957), whereas that of the C4 was
cillata leaves in the C3 state and during induction of the C4 state. One sigmoid and fitted the Hill equation (r2 ⫽ 0.998). The
microgram per lane of total RNA from H. verticillata C3 and C4 leaves
Hill coefficients for the two extracts differed consid-
was separated on a 1.2% (w/v) denaturing agarose gel and blotted
onto a positively charged nylon membrane. DIG-labeled 3⬘-end RNA
erably, 1.8 and 3.8 for the C3 and C4 leaves, respec-
probes from Hvpepc3 and Hvpepc4, and a consensus probe were tively. The specific activities, calculated from the
used to hybridize the membranes for transcript identification. The Michaelis-Menten and Hill equations, were several-
ethidium bromide-stained gel shows uniform loading of RNA sam- fold different, with the C4 value the higher (2.51
ples. The size (kb) of the full-length cDNAs encoding PEPC isoforms versus 0.37 mol mg⫺1 protein min⫺1). In contrast,
is indicated on the right. the K0.5 PEP values did not differ substantially,
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.Rao et al.
Figure 3. Specific activities of PEPC in desalted
extracts of H. verticillata leaves in the C3 state
and during induction of the C4 state. The PEPC
was extracted from leaves harvested during the
light period, and activity was measured with
saturating substrates at pH 8. The arrows indi-
cate sampling times for the northern analyses.
Data are means ⫾ SE, n ⫽ 3.
whether estimated from the graph or calculated by with a set of two or more isoforms, so that diversity
the Hill equation, and in addition, they were rela- of isoform function was represented. Using the
tively high (Fig. 4). PHYLIP or the PAUP package (Phylogenetic Analy-
Western analyses showed two prominent immuno- sis Using Parsimony, version 4.0, Sinaur Associates,
reactive bands in both leaf extracts, with the second Sunderland, MA), both the protein distance and pro-
being much more pronounced in the C4 leaves (Fig. tein parsimony methods gave consensus trees that
5). In addition, a third, faster running band was were very similar. For these analyses, the four pro-
evident only in the C4 leaf extract. The distribution of karyotic sequences were taken as the outgroup,
these bands was in the Mr range of 105,000 to 111,000. showing similarity with the seed plant sequences in
the range of 26% to 39%. In all, 943 total characters
Phylogenetic Analyses were considered, and 608 of them were parsimony
informative. The consistency and retention indices
Figure 6 shows the results of a phylogenetic anal- were 0.71 and 0.63, respectively, indicating low ho-
ysis of deduced amino acid sequences with the moplasy or background noise because of conver-
PHYLIP program (Phylogeny Inference Package, ver- gence or reversion events. The root PEPC isoforms
sion 3.57c, Department of Genetics, University of of the graminaceous plants; maize, sorghum (Sor-
Washington, Seattle) using the parsimony algorithm. ghum vulgare), and rice (Oryza sativa); and the C4
In addition to the three H. verticillata PEPC se- sequences of maize and sorghum apparently di-
quences, 28 other full-length sequences from Gen- verged independently.
Bank representing 17 different taxa were included. From this analysis, it appeared that PEPC isoforms
Particular emphasis was placed on selecting species can be grouped into three distinct groups that likely
Table II. Partial purification of PEPC extracted from H. verticillata C3 and C4 leaves and roots
Leaves of H. verticillata were harvested midway through their light cycle, C3 at 0 h and C4 at 288 h into
the induction period. The PEPC of C3 and C4 leaves eluted as single peaks but at different 关KCl兴.
Ammonium sulfate precipitation was not employed for root extracts because of the initial low activity. The
protein from the root extract eluted in the buffer wash, and did not bind to the Sepharose column. Specific
activity was assayed with saturating substrate concentrations at pH 8. N.D., Not detemined.
Specific Activity
Peak Elution Purification
Source Peak Recovery
Crude (NH4)2SO4 Pellet Concentration Factor
Fraction
mol mg⫺1 protein min⫺1 mM %
C3 leaves 0.078 0.182 0.590 200 7.6 62
C4 leaves 0.589 0.743 2.830 262 4.8 44
Root 0.030 N.D. 0.080 0 2.7 66
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.Phosphoenolpyruvate Carboxylase Isoforms in Hydrilla verticillata
Figure 4. The effect of PEP concentration on the
specific activity of PEPC in the C3 and C4 leaf
peak fractions from anion-exchange chromatog-
raphy (see Table II). The PEPC was extracted
from the H. verticillata leaves harvested during
the light period. The C4 sample was taken 288 h
into the induction period. The assay was run at
pH 7.3 in the absence of dithiothreitol. The
apparent K0.5 PEP values were determined from
the curves. Data are means ⫾ SE, n ⫽ 3.
share a common ancestor: I, C4 graminaceous; II, pea (Pisum sativum) and the H. verticillata sequences
graminaceous roots; and III, PEPC isoforms with vary- appear to form a clade that is present regardless of tree
ing functions from a variety of taxa. Although group construction methods.
III was monophyletic, relationships within it were
largely unresolved because the component branches
lacked statistical support. Nonetheless, there was DISCUSSION
good support for several clades, namely Brassica spp.,
Flaveria spp., Hydrilla spp., and a Sorghum spp./Zea Photosynthesis in H. verticillata is unique in that,
spp. group (C3 PEPC). Within the genus Flaveria, the against a C3 background, a C4 cycle is induced but
C3 species Flaveria pringlei ppcA showed a clear diver- without the development of specialized anatomy that
gence from the C4 F. trinervia ppcA, with 100% support. occurs in terrestrial C4 species. This “minimalist”
However, the sequences from both the C3 and C4 system represents something of a paradox in our
Flaveria spp. fell into the same group as those from H. concept of C4 photosynthesis. Since the classical C3 ⫻
verticillata. In the case of H. verticillata, Hvpepc3 and C4 Atriplex spp. hybridization experiments of Björk-
Hvpepc5, and Hvpepc4 diverged from a unique com- man et al. (1970), it has been accepted that for a C4
mon C3 ancestor. It is intriguing that the gymnosperm system to concentrate CO2 and to avoid its futile
Norway spruce (Picea abies) along with the root nodule cycling, the biochemical components need to be seg-
regated in specific cell types. H. verticillata was the
only exception, but others have been reported re-
cently (Bowes et al., 2002), including Borszczowia
aralocaspica, a terrestrial NAD-ME C4 species in
which the C4 and Calvin cycles appear to co-exist in
different regions of the same cell (Voznesenskaya et
al., 2002).
The inducible H. verticillata system provides an
excellent opportunity to study the minimum essen-
tial biochemical elements to operate a C4 photosyn-
thetic system, such as might be needed to transform
a C3 crop plant. Its facultative nature also enables us
to examine the genes involved in both the C3 and C4
states, differences in their expression, and variations
Figure 5. Western analyses of PEPC from leaves of H. verticillata. in the regulatory and catalytic domains of their
Leaves of H. verticillata were harvested midway through their light products.
cycle, C3 at 0 h and C4 at 288 h into the induction period. Six
micrograms of protein from the C3 and C4 leaf peak fractions from
We have previously described the major physiolog-
anion-exchange chromatography (see Table II) was resolved by 5% ical and biochemical features of the system (Bowes
(w/v) SDS-PAGE and transblotted to a nitrocellulose membrane. The and Salvucci, 1989; Magnin et al., 1997). Thus, the
membrane was probed with antibodies raised against maize PEPC. purpose of this study was to begin to elucidate the
The PEPC signals from C3 and C4 leaves are shown. The kilodalton molecular mechanisms involved, particularly those
value of the prominent PEPC band is indicated at the right. associated with the induction and role(s) of PEPC,
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.Rao et al.
Figure 6. Phylogenetic analysis of deduced amino acid PEPC sequences. The PHYLIP package was used to construct a
consensus tree with 100 bootstrap replications using the parsimony method. The four prokaryotic species served as the
outgroup. The stars at the fork of the tree represent ⬎85% support. The three groupings are: I, C4 graminaceous; II,
graminaceous roots; and III, sequences of other higher plants. Deduced amino acid sequences other than H. verticillata were
obtained from GenBank/SwissProt.
the first element in the C4 pathway. The genes en- the C3 (non-photosynthetic forms), C4, and CAM iso-
coding PEPC isoforms in terrestrial plants have been forms (Lepiniec et al., 1993; Ernst and Westhoff, 1997;
well described, and distinctions can be made among Svensson et al., 1997; Cushman and Bohnert, 1999).
882 Plant Physiol. Vol. 130, 2002
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.Phosphoenolpyruvate Carboxylase Isoforms in Hydrilla verticillata
Of the three PEPC isoforms from H. verticillata form is undetermined. However, PEPC operating in a
Hvpepc4 was expressed solely in C4 leaves. Several C4 CCM may need enhanced capacity to respond
lines of evidence point to this isoform as the photo- rapidly as metabolites fluctuate with transient
synthetic PEPC operating in the light. It was only changes in the environment.
isolated from C4 leaf RNA and was only expressed in The expression pattern and kinetic data point to
C4 leaves, and its expression coincided with the rise Hvpepc4 as the C4 photosynthetic PEPC. What then is
in PEPC activity as the C4 system was induced. In the role for Hvpepc3 in the leaves? H. verticillata
addition, its sequence least resembled those of leaves can fix CO2 in the dark, at 12% of the light rate
Hvpepc3 or Hvpepc5 that were isolated from C3 leaves in the case of C4 leaves, and they accumulate malate
and roots, respectively. It also contained the F. tri- (Reiskind et al., 1997; J.B. Reiskind, S.K. Rao, and G.
nervia C4 Lys-347, though as noted earlier, this resi- Bowes, unpublished data). The ability of a PEPC that
due is not a very specific determinant of a C4 PEPC is not light-regulated to scavenge inorganic carbon at
isoform. The C4-signature Ser residue was absent night when concentrations rise could be another fac-
from all H. verticillata sequences, and instead, Ala, tor in the plant’s carbon economy in habitats where
which is typical of C3 sequences, occurred at this dissolved CO2 becomes a major daytime limitation.
position. Ser appears to be ubiquitous at this position The sequence similarity between Hvpepc3 and 5
among the C4 isoforms of terrestrial C4 plants, and it might suggest that the same gene encodes them both.
plays a role in determining the kinetic characteristics However, this is unlikely because all of the 3⬘-UTR
(Bläsing et al., 2000). How the H. verticillata PEPC sequences analyzed to date from independent clones
functions kinetically as a C4 photosynthetic isoform of three organ sources, i.e. leaf, root, and subterra-
with the C3 Ala at this position, instead of Ser, is an nean and axillary turions, revealed (a) three distinct
interesting issue that deserves further study. 3⬘-UTR categories; (b) that the Hvpepc5-like se-
We recently reported that PEPC in desalted ex- quences were the same length and were 99% homol-
tracts from C3 and C4 H. verticillata leaves differed ogous; and (c) that a specific polyadenylation signal
kinetically in that the C4 leaf enzyme is light acti- site at a common position (nt 3,198 to 3,203 in
vated and is over 10-fold more sensitive to malate Hvpepc3 and Hvpepc5) was present.
inhibition (Bowes et al., 2002). In the present study, The three full-length cDNA H. verticillata sequences
the specific activity of C4 leaf PEPC was substantially were all very similar (95%–99%). A comparable situ-
higher than that from C3 leaves. Among terrestrial ation is seen in Kalanchoe blossfeldiana where two
plants, PEPC in C4 leaves is light-activated, and its pairs of isogenes encode highly similar C3- and
activity is similarly severalfold higher than that from CAM-specific PEPC isoforms, with the slight devia-
C3 leaves when assayed at a cytosolic-like pH (Gupta tions being attributed to gene duplication or the hy-
et al., 1994). brid status of the plant in which the parental ge-
The Km PEP values for PEPC differ among terres- nomes are expressed (Gehrig et al., 1995). Gene
trial plant C3 and C4 enzymes (O’Leary, 1982). This, duplication could be the case in H. verticillata, be-
however, was not the case for PEPC from C3 and C4 cause the plants in Florida are dioecious female dip-
H. verticillata leaves, which had similar K0.5 PEP val- loids (2n ⫽ 16) and are materlineal (Langeland et al.,
ues that were high and C4-like, confirming much 1992). Variable length and base pair differences of the
earlier measurements with crude extracts (Nakamura UTRs, particularly at the 3⬘ end where message sta-
et al., 1983). Bläsing et al. (2000) showed that in bility is an issue, may determine functional proper-
site-directed mutagenesis and chimeric constructs of ties of encoded proteins (Ingelbrecht et al., 1989).
ppcA PEPC from C3 F. pringlei and C4 F. trinervia, the These could be elements governing functional differ-
replacement of Ala-774 with Ser increases the K0.5 ences among the H. verticillata isoforms. In addition,
PEP of the recombinant proteins. They concluded there were two initiation codons downstream of the
that Ser-774 is a key determinant of C4-like kinetics, leader sequence, which are seen in other PEPC se-
including a high K0.5 PEP. Thus, the similarity of H. quences (Relle and Wild, 1996). If translation is initi-
verticillata K0.5 PEP values might be expected, be- ated from the second Met, then the motif upstream of
cause the sequences are identical at this site. In con- the Ser residue is absent and the interaction of this
trast, the presence of Ala and high K0.5 values does residue with PEPC-protein kinase and the subse-
not support a ubiquitous need for Ser at this position quent phosphorylation would not occur.
to obtain a C4-like K0.5 PEP. All but two plant PEPCs in GenBank contain a Cys
Hill coefficients for recombinant ppcA PEPCs from residue at position 196, but Ser occurred in Hvpepc5.
C3 and C4 Flaveria spp. indicate the C4 enzyme has At 891, Arg is the residue most commonly found, and
greater positive cooperativity (Bläsing et al., 2000). it was conserved in Hvpepc5, but in both Hvpepc3 and
The H. verticillata data parallel this, in that the C4 leaf Hvpepc4, Glu was substituted. The Met-150 in
PEPC was strongly homotropic with PEP acting as a Hvpepc4 was also unusual, because the conserved
positive modulator. A similar situation exists with residue is Leu. It is not clear whether these diver-
maize (Tovar-Mendez et al., 1998). The in vivo role gences influence the kinetic and regulatory charac-
for allosteric regulation of the C4 photosynthetic iso- teristics of the isoforms. As noted earlier, the absence
Plant Physiol. Vol. 130,Downloaded
2002 from www.plantphysiol.org on September 16, 2015 - Published by www.plant.org 883
Copyright © 2002 American Society of Plant Biologists. All rights reserved.Rao et al.
of the C4 signature Ser is a very unusual feature of (Magnin et al., 1997). Induction was followed over time by determining the
increase in PEPC activity, and leaves in the C3 and C4 state were harvested
the H. verticillata photosynthetic PEPC sequence.
at intervals. Rooting of H. verticillata sprigs was achieved by planting them
The deduced amino acid sequences of the three in sand under a 12-h 25°C photoperiod/25°C scotoperiod. Roots were
full-length PEPC isoforms indicated that they had harvested 3 or 4 weeks after planting.
similar pIs and Mrs. This may be why Q-Sepharose
chromatography of C4 leaf extracts did not yield two
PEPC Assay, Western-Blot Analyses, and
peaks, even though northern analyses showed the
Protein Purification
presence of two isoforms. Of the immunoreactive
bands resolved on SDS-PAGE, only the second cor- Enzyme activities for maximal activity and western blots were performed
responded with the deduced Mr of the three identi- as previously described (Magnin et al., 1997). For the latter, polyclonal
antibodies raised against maize PEPC were used. K0.5 PEP values and
fied isoforms. The others may be cross-reacting pro- maximal velocities were assessed at pH 7.3 in the absence of dithiothreitol
teins or other isoforms. Similar banding patterns for with [PEP] ranging from 0 to 2 mm. Protein was determined by the Bradford
PEPC have been observed in Egeria spp. and Sorghum method with ␥-globulin as the standard (Bradford, 1976). PEPC was purified
spp. with the conclusion that they represented differ- by (NH4)2SO4 fractionation (25%–55% [w/v]) followed by desalting on
ent PEPC isoforms (Casati et al., 2000; Nhiri et al., PD-10 columns (Amersham Biosciences AB, Uppsala) equilibrated with 20
mm PIPES, pH 7.0, 10 mm MgCl2, 10% (v/v) glycerol, and 10 mm
2000). -mercaptoethanol. The resulting eluate was applied to a 1-mL Q-Sepharose
The phylogenetic analyses indicated that the H. FF column (Amersham Biosciences AB) equilibrated with running buffer
verticillata sequences, including Hvpepc4, were diver- (RB; 20 mm PIPES, pH 7.0, and 10 mm -mercaptoethanol). After a RB wash,
gent from the C4 graminaceous PEPCs. The C4 F. the bound protein was eluted with a 30-mL linear KCl gradient (0–400 mm)
in RB and collected in 0.5-mL fractions for PEPC assay.
trinervia PEPC similarly grouped with C3 and CAM
PEPCs from monocots and eudicots. Thus, the func-
tional diversity of PEPC isoforms was not fully re- Cloning and cDNA Sequencing
flected in the branching pattern. It is possible that the
Total RNA was extracted from C4 leaves, roots, subterranean and axillary
C4 form of PEPC diverged before the monocot/ turions (Qiagen RNeasy Kit, Qiagen USA, Valencia, CA), and RACE-ready
eudicot split 200 million years ago (mya) but after the cDNA was prepared from it using the SMART RACE cDNA Amplification
gymnosperm and angiosperm divergence 330 mya Kit (BD Biosciences Clontech, Palo Alto, CA). PEPC-specific primers 8F
(Wolfe et al., 1989; Relle and Wild, 1996). The PEPC (5⬘-GCGAAGCAATATGGAGTGAAGTTGA-3⬘; corresponding to nt 79–103)
and 11R (5⬘-TTGTACATTGTACCCTGGGTCCCTT-3⬘; nt 933–957) were de-
from Norway spruce, which is suggested to be part of signed from the partial cDNA sequence Hvpepc2 obtained previously (Rao et
the N-fixation system in spruce roots (Relle and al., 1998). TA cloning of the RACE products was performed with the
Wild, 1996) and, thus, is likely related to the pea root TOPO-XL PCR Cloning Kit (Invitrogen, Carlsbad, CA). PEPC-specific in-
nodule PEPC, was potentially a sister to the H. verti- serts were initially confirmed by screening with the DIG-oligo tailed primer
11R (Roche Diagnostics/Roche Applied Science). From partial sequencing
cillata isoforms and was closer to them than to other
of the 5⬘ end of several of these clones, two primers, 3F (5⬘-CGCGTCTGT
monocot C3 or CAM PEPCs. If so, the H. verticillata TCTGATGGCGTC-3⬘; nt 47–67) and 4F (5⬘-TGCGCGAGTGTCCCGATGG3⬘;
PEPCs may represent ancestral sets of genes that nt 47–65), were designed and DIG-tailed to aid in further screening. Clones
emerged before angiosperm divergence and may from this screening were partially sequenced to identify the extreme 5⬘- and
provide clues to C4 evolution in monocots. It should 3⬘-cDNA ends, so that specific primers could be designed to amplify full-
length cDNAs encoding PEPC isoforms. A primer walking strategy was
be noted that monocot PEPC genes may have di- used for sequencing. The full-length sequence data reported here are in the
verged early into the C4 type and were not necessar- GenBank at the National Center for Biotechnology Information under the
ily accompanied by C4 photosynthesis (Kawamura et accession numbers AF271161 (Hvpepc3), AF271162 (Hvpepc4), and AF271163
al., 1992). (Hvpepc5).
Members of the Hydrocharitaceae, to which H. ver-
ticillata belongs, were adapted to an aquatic environ- Northern Analyses
ment 120 mya (Sculthorpe, 1967). Aquatic habitats
may experience very low daytime CO2 to O2 ratios, For northern analyses, a total of 1 g of RNA per lane, extracted from
leaves (RNeasy Plant Kit, Qiagen USA), was separated on a 1.2% (w/v)
particularly in heavily vegetated areas (Bowes and agarose formaldehyde gel (Maniatis et al., 1982). A downward capillary
Salvucci, 1989), so submersed species likely experi- blotting method was employed to transfer the RNA to a positively charged
enced low [CO2] before terrestrial plants encountered nylon membrane using 10⫻ SSC as the transfer buffer (Roche Diagnostics/
such conditions. Some submersed species show evi- Roche Applied Science). The bound RNA was UV cross-linked for 3 min and
hybridized overnight with the appropriate DIG-labeled RNA probe in stan-
dence of C4 photosynthesis (Bowes et al., 2002), and
dard hybridization buffer with 50% (v/v) formamide. The stringency
it is possible an early selection pressure led to its washes and detection were carried out following the DIG-System User’s
presence in submersed species, like H. verticillata, Guide (Roche Diagnostics/Roche Applied Science). For stripping the probes
before its advent in terrestrial plants. from the hybridized membranes, two washes at 80°C for 1 h each were
performed with 50% (v/v) formamide and 5% (w/v) SDS in 50 mm Tris-HCl
at pH 7.5.
MATERIALS AND METHODS
Plant Material Syntheses of Antisense RNA Probes
Hydrilla verticillata (L.f.) Royle sprigs 6 cm long were incubated with a Three different antisense RNA probes were synthesized following the
photon irradiance of 300 mol m⫺2 s⫺1 under a 14-h 30°C photoperiod/ protocol of the DIG RNA labeling kit (Roche Diagnostics/Roche Applied
22°C scotoperiod to limit daytime [CO2] and induce C4 photosynthesis, or a Sciences). PCR amplified regions from either full- or partial-length cDNA
10-h 15°C photoperiod/10°C scotoperiod regime to maintain the C3 state clones were inserted into the vector pCR-XL-TOPO (Invitrogen) in a manner
884 Plant Physiol. Vol. 130, 2002
Downloaded from www.plantphysiol.org on September 16, 2015 - Published by www.plant.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.Phosphoenolpyruvate Carboxylase Isoforms in Hydrilla verticillata
such that the transcription template included the T7 promoter/priming site at Bowes G, Salvucci ME (1989) Plasticity in the photosynthetic carbon me-
the 3⬘ end. The specific probes for Hvpepc3 (nt 2,948–3,368) and Hvpepc4 (nt tabolism of submersed aquatic macrophytes. Aquat Bot 34: 232–266
2,944–3,197) were derived from their respective full-length cDNA clones with Bradford MM (1976) A rapid and sensitive method for the quantification of
the primer pairs PRB-3P (5⬘-TGCTGGCATGCAGAACACTGGTTAACC-3⬘) microgram quantities of protein utilizing the principle of protein-dye
and T7-PCR primer (5⬘-TAATACGACTCACTATAGGG-3⬘). The region (nt binding. Anal Biochem 72: 248–254
47–1,799) of the consensus probe was PCR amplified from a 1.8-kb partial Casati P, Lara MV, Andreo CS (2000) Induction of a C4-like mechanism of
cDNA clone of Hvpepc3 with the aid of primer pairs 3F (5⬘-CGCGTCTGT- CO2 fixation in Egeria densa, a submersed aquatic species. Plant Physiol
TCTGATGGCGTC-3⬘) and T7-PCR primer. 123: 1611–1622
Chollet R, Vidal J, O’Leary MH (1996) Phosphoenolpyruvate carboxylase: a
ubiquitous highly regulated enzyme in plants. Annu Rev Plant Physiol
Sequence Analyses and Phylogeny Inference Plant Mol Biol 47: 273–298
Cushman JC, Bohnert HJ (1999) Crassulacean acid metabolism: molecular
Standard sequence compiling and analyses, including pair-wise compar-
genetics. Annu Rev Plant Physiol Plant Mol Biol 50: 305–332
ison of nt and deduced amino acids, were performed using the Wisconsin
Ernst K, Westhoff P (1997) The phosphoenolpyruvate carboxylase (ppc)
package (v10.1, Genetics Computer Group, Madison, WI). For phylogenetic
gene family of Flaveria trinervia (C4) and F. pringlei (C3): molecular char-
analysis, PHYLIP v3.57 (Felsenstein, 1989) and PAUP programs were used.
acterization and expression analysis of the ppcB and ppcC genes. Plant
The deduced amino acid sequences of the three full-length H. verticillata
Mol Biol 34: 427–443
PEPC isoforms and 28 previously published PEPC sequences from GenBank
Felsenstein J (1989) PHYLIP: phylogeny inference package (version 3.2).
were used to build the tree. The species and accession numbers of the 28
Cladistics 5: 164–166
PEPC sequences are: Anacystis nidulans (M11198), Anabaena variabilis
Gehrig H, Taybi T, Kluge M, Brulfert J (1995) Identification of multiple
(M80541), Norway spruce (Picea abies; X79090), pea (Pisum sativum; D64037),
isogenes in leaves of the facultative Crassulacean acid metabolism (CAM)
rice (Oryza sativa; AF271995), tobacco (Nicotiana tabacum; X59016), common
plant Kalanchoe blossfeldiana Poelln. cv. Tom Thumb. FEBS Lett 377:
ice plant (Mesembryanthemum crystallinum; ppc2, X14588; ppc1, X14587),
399–402
Vanilla planifolia (ppcV1, X87148; ppcV2, X87149), maize (Zea mays; C3,
Gupta SK, Ku MSB, Lin J-H, Zhang D, Edwards GE (1994) Light/dark
X61489; root, AB012228; C4, X03613), sorghum (Sorghum vulgare; CP21,
X63756; CP46, X65137; CP28, X59925), Flaveria trinervia (ppcA, X64143; ppcB, modulation of phosphoenolpyruvate carboxylase in C3 and C4 species.
AF248079; ppcC, AF248080), Flaveria pringlei (ppcA, Z48966), sugarcane Photosynth Res 42: 133–143
(Saccharum officinarum; C4, AJ293346), Saccharum hybrid var H32–8560 (C3, Hermans J, Westhoff P (1992) Homologous genes for the C4 isoform of
M86661), tomato (Lycopersicon esculentum; ppc1, AJ243416; ppc2, AJ243417), phosphoenolpyruvate carboxylase in a C3 and a C4 Flaveria species. Mol
brown mustard (Brassica juncea; ppc2, AJ223496; ppc3, AJ223497), Strepto- Gen Genet 234: 275–284
myces coelicolor (CAB95920), and Escherichia coli (AE000469). Ingelbrecht ILW, Herman LMF, Dekeyser RA, Van Montagu MC, De-
The predicted protein sequences were aligned using the CLUSTAL pro- picker AG (1989) Different 3⬘ end regions strongly influence the level of
gram (Thompson et al., 1994), and the sequences were edited to include only gene expression in plant cells. Plant Cell 1: 671–680
the unambiguously aligned sections. Two different methods in the PHYLIP Kawamura T, Shigesada K, Toh H, Okumura S, Yanagisawa S, Isui K
package, NEIGHBOR (neighbor-joining based on the output file from PRO- (1992) Molecular evolution of phosphoenolpyruvate carboxylase for C4
TDIST distance matrix analysis program) and PROTPARS (maximum parsi- photosynthesis in maize: comparison of its cDNA sequence with a newly
mony), were used with a bootstrap analysis of 100 replications to determine isolated cDNA encoding an isozyme involved in the anapleurotic func-
and compare the confidence level of branches within the phylogenetic tree. tion. J Biochem 112: 147–154
Kellogg EA (1999) Phylogenetic aspects of the evolution of C4 photosyn-
thesis. In RF Sage, RK Monson, eds, C4 Plant Biology. Academic Press,
Distribution of Materials San Diego, pp 411–444
Ku MSB, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose
Upon request, all novel materials described in this publication will be S, Toki S, Miyao M, Matsuoka M (1999) High-level expression of maize
made available in a timely manner for noncommercial research purposes, phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotech-
subject to the requisite permission from any third-party owners of all or nol 17: 76–80
parts of the material. Obtaining any permissions will be the responsibility of Langeland KA, Shilling DG, Carter JL, Laroche FB, Steward KK, Madiera
the requestor. PT (1992) Chromosome morphology and number in various populations
of Hydrilla verticillata. Aquat Bot 42: 253–263
Lepiniec L, Keryer E, Philippe H, Gadal P, Cretin C (1993) Sorghum
ACKNOWLEDGMENTS phosphoenolpyruvate carboxylase gene family: structure, function and
molecular evolution. Plant Mol Biol 21: 487–502
We thank Dr. Michael Salvucci of the U.S. Department of Agriculture-
Lepiniec L, Vidal J, Chollet R, Gadal P, Cretin C (1994) Phosphoenolpyru-
Agricultural Research Service, Western Cotton Research Laboratory (Tuc-
vate carboxylase: structure, regulation and evolution. Plant Sci 99:
son, AZ) for the generous gift of the antibody to PEPC from maize. We also
111–124
thank Drs. Walter Judd and Mark Whitten of the University of Florida
Magnin NC, Cooley BA, Reiskind JB, Bowes G (1997) Regulation and
Department of Botany (Gainesville, FL) and the Florida Museum of Natural
localization of key enzymes during the induction of Kranz-less, C4-type
History (Gainesville, FL), respectively, for their advice on phylogenetic tree
photosynthesis in Hydrilla verticillata. Plant Physiol 115: 1681–1689
construction.
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: A Laboratory
Received May 7, 2002; returned for revision May 28, 2002; accepted June 13, Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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