o
n
,?
*Sm 094
rm
ed
To b
phosp
its ver
and ta
compa
acid s
gene)
of Pte
with p
quenc
compl
sequen
positio
functi
furthe
provid
tion o
metab
cussio
tation
Key
photo
lar e
(PEPC
The comparison of organisms on the level of molec-
ular ch
pensa
Howev
this ap
versat
four t
marke
sius, 1
Bruns
box ge
coding
C
9).
t
er
ly
no
rch
ed
PC
EP
uv
ro
19
roo
cti
2 fi
hm
du
tio
a
e m
ze
ie
we
sh
tsc
al., 1993, 1992; Gehrig et al., 1995) encoding function-
and tissue-specific isoforms of the enzyme (Lepiniec et
1 To
dressed
Molecular Phylogenetics and Evolution
Vol. 20,
doi:10.1 on
1055-790
Copyrigh
All rightaracters has become a powerful and now indis-
ble tool in taxonomic and phylogenetic research.
er, the unequivocalness of results obtained by
proach depends essentially on the availability of
ile molecular markers. In plant sciences mainly
ypes of nucleotide sequences are used as such
rs, namely the 18s rRNA (e.g., Bopp and Cape-
996; Qiu and Palmer, 1999), ITS regions (e.g.,
al., 1994; Toh et al., 1994; Rajagopalan et al., 1994;
Gehrig et al., 1998b). Because of the ubiquitous distri-
bution of PEPC and the high diversity in its functions
it has been proposed that the nucleotide sequences of
the PEPC genes and the amino acid sequences of the
gene products should provide powerful markers in
molecular taxonomic and phylogenetic investigations
(Gehrig et al., 1998a).
First attempts to construct phylogenetic trees ofNew Partial Sequences of Phosph
as Molecular Phyloge
Hans Gehrig,* ,1 Valentina Heute
ithsonian Tropical Research Institute, Unit 0948, APO AA 34002-
Schnittspahnstr. 10, 64287 Da
Received October 10, 2000; revis
etter understand the evolution of the enzyme
hoenolpyruvate carboxylase (PEPC) and to test
satility as a molecular character in phylogenetic
xonomic studies, we have characterized and
red 70 new partial PEPC nucleotide and amino
equences (about 1100 bp of the 3* side of the
from 50 plant species (24 species of Bryophyta, 1
ridophyta, and 25 of Spermatophyta). Together
reviously published data, the new set of se-
es allowed us to construct the up to now most
ete phylogenetic tree of PEPC, where the PEPC
ces cluster according to both the taxonomic
ns of the donor plants and the assumed specific
on of the PEPC isoforms. Altogether, the study
r strengthens the view that PEPC sequences can
e interesting information for the reconstruc-
f phylogenetic relations between organisms and
olic pathways. To avoid confusion in future dis-
n, we propose a new nomenclature for the deno-
of PEPC isoforms. ? 2001 Academic Press
Words: crassulacean acid metabolism (CAM);
synthesis (C3, C4); molecular taxonomy; molecu-
volution; phosphoenolpyruvate carboxylase
).
INTRODUCTION
and
199
men
trov
high
taxo
Sea
par
(PE
P
pyr
as p
al.,
mic
fun
CO
Cus
pro
fixa
stom
stat
Mel
stud
sho
(Cu
Poe
No. 2, August, pp. 262?274, 2001
006/mpev.2001.0973, available online at http://www.idealibrary.comet al., 1991; Bogler and Simpson, 1996), MADS-
nes (e.g., Winter et al., 1999), and the rbcL genes
for the large subunit of RUBISCO (e.g., Dressler
PEPC
al., 19
1996;
that t
provid
whom correspondence and reprint requests should be ad-
. Fax: (00507)-212-8148. E-mail: hansgehrig@gmx.de.
3/01 $35.00
t ? 2001 by Academic Press
s of reproduction in any form reserved.
262enolpyruvate Carboxylase
etic Markers
and Manfred Kluge?
8, Panama; and ?Darmstadt University of Technology,
stadt, Germany
March 12, 2001
hase, 1995; Yukawa et al., 1996; Qiu and Palmer,
Because the obvious limitation in the assort-
of suitable markers may be one reason for con-
sial interpretations of obtained results, it is
desired that more markers become available for
mic and phylogenetic studies in plant sciences.
ing for such markers, we investigated and com-
sequences of phosphoenolpyruvate carboxylase
; EC 4.1.1.31).
C catalyzes the b-carboxylation of phosphoenol-
ate, with oxaloacetate and inorganic phosphate
ducts (Utter and Kolenbrander, 1972; Andreo et
87). The enzyme is ubiquitous in prokaryotic
rganisms and plants, and it is involved in many
ons including photosynthetic and anaplerotic
xation (e.g., Kluge and Ting, 1978; Winter, 1985;
an and Bohnert, 1999; Latzko and Kelly, 1983),
ction of carbon skeletons in symbiotic nitrogen
n (Schuller et al., 1990), modulation of turgor in
tal guard cells, maintenance of ion balance, pH
echanisms, and others (Latzko and Kelly, 1983;
r and O?Leary, 1987). In most bacteria and plants
d so far, physiological and molecular approaches
d the existence of PEPC multigene families
man and Bohnert, 1989a,b; Cre?tin et al., 1991;
h et al., 1991; Kawamura et al., 1992; Lepiniec etwere based on full-length sequences (Lepiniec et
93; Toh et al., 1994; Cushman and Bohnert,
Honda et al., 1996). Gehrig et al. (1998a) showed
he comparison of partial PEPC sequences can
e valuable information on the phylogenetic in-
terrela
plants
up to
sequen
alga (2
and 25
determ
Gehrig
phylog
cies is
compl
sequen
Bryop
presen
Plant
The
tanica
many)
with s
ter th
were i
at 280
RNA E
Tota
cyana
QIAGE
depen
exami
verse
was am
with
(CTA)
PEPC
AT(GT
(Perso
55?C f
cloned
erland
choe a
with B
lyzed
Ran
PEPC
(SeqLa
quenc
GenBa
for acc
Seque
Ami
otide
identi
ment
ed
to
me
ste
N
OT
u
L
u
su
en
m
he
ed
he
s
s a
od
ed
re
C
orm
, d
C
ing
ble
the
ph
R 5
p
2 fi
m
)
t
orm
4;
vo
C3
wi
t
e
log
e a
r
cie
uen
ed
ge
nc
e b
cie
cie
263PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKERtionships of PEPC isoforms and the donor
from which the genes were isolated. Although
now full-length and partial PEPC nucleotide
ces of 11 prokaryotes (11 PEPC sequences), 1
PEPC sequences), 5 ferns (5 PEPC sequences)
higher plants (48 PEPC sequences) have been
ined (Toh et al., 1994; Chollet et al., 1996;
et al., 1998a) our present knowledge on the
eny of PEPC sequences and the isoforms in spe-
still quite fragmentary and urgently requires
etion. For this reason we have analyzed PEPC
ces in numerous further plant species including
hyta, Pteridophyta, and Spermatophyta. The
t paper reports on the obtained results.
MATERIALS AND METHODS
Material
experimental plants were cultivated in the Bo-
l Gardens of Heidelberg and Darmstadt (Ger-
. The plant material was thoroughly cleaned
terilized water and by ultrasonic treatment. Af-
e cleaning, tissue samples for RNA preparation
mmediately frozen in liquid nitrogen and stored
?C until further processing.
xtraction and PCR Amplification
l RNA was extracted with the guanidine isothio-
te method (Chirgwin et al., 1979) or with the
N plant RNA isolation kit (Qiagen, Germany),
ding on the plant material. RNA quality was
ned by agarose gel electrophoresis, and after re-
transcription an approx 1100-bp PEPC fragment
plified by RT-PCR. The RT-PCR was performed
two degenerated PEPC primers [PEPC1: TC-
GA(TC) TC(CAT) GG(AC) AA(AG) GA(TC) GC;
2: GC(GAT) GC(GAT) AT(GCA) CC(CT) TTC
) G] under the following conditions: 35 cycles
nal Cycler; Biometra, Germany) at 95?C for 30 s,
or 30 s, 72?C for 3 min. The PCR products were
into the TA vector system of Invitrogen (Neth-
s). Different PEPC isoform clones of the Kalan-
nd orchid species were identified by digestion
amHI, HindIII, PstI, SalI, and EcoRI and ana-
on 0.8% agarose gels.
domly selected transformants of each amplified
cDNA clone were sequenced in both directions
b Co., Hannover, Germany). The nucleotide se-
e data have been submitted to the EMBL and
nk nucleotide sequence databases (see Table 1
ession numbers).
nce Analysis
no acid sequences were obtained from 143 nucle-
PEPC sequences. The alignment and sequence
ty were calculated for each pair with the Align-
tain
the
from
by
sen
and
(PR
form
PHY
form
mea
twe
resa
of t
root
furt
case
A
is c
link
the
PEP
isof
sion
PEP
dist
(Ta
syn
non
for
and
CO
acid
CAM
ture
isof
199
be a
the
we
In
serv
phy
hav
The
spe
seq
pos
Alto
que
gen
spe
speEditor 3.7 (Hepperle, 1997). The alignment ob- Aswas modified by visual inspection to increase
tal alignment score (the alignment is available
the authors). Sequence data were evaluated
ans of the PHYLIP package, version 3.5c (Fel-
in, 1993). Neighbor-joining analysis (Saitou
ei, 1987) was employed as a distance method
DIST) with 1000 resamplings with the Kimura
la for amino acid sequences (Kimura, 1983) of the
IP package. This is a rough-and-ready distance
la for approximating PAM distance that simply
res the fraction of amino acids that differs be-
two sequences. Parsimony analyses with 100
plings were done with the PROTPARS program
PHYLIP package. This program infers an un-
phylogeny directly from protein sequences (for
r explanations see Felsenstein, 1993). In all
Escherichia coli was used as outgroup.
RESULTS AND DISCUSSION
lready mentioned in the Introduction, the PEPC
ed by multigene families, with isoforms being
to a wide range of different functions. Up to now
has been no generally followed terminology of
sequences suitable for relating a given PEPC
to a specific function. However, to avoid confu-
efinition of a common nomenclature to denote
isoforms is highly desired. Thus, we propose to
uish and to denote PEPC isoforms as follows
2): prokaryotic anaplerotic and other nonphoto-
tic isoforms (ppc-aP), eukaryotic anaplerotic and
otosynthetic isoforms (ppc-aX; with X standing
root, aerial root, root nodule, and for L 5 leaf),
hotosynthetic isoforms catalyzing the primary
xation in C4 photosynthesis and crassulacean
etabolism (CAM) (respectively, ppc-C4 and ppc-
. The term ?C3? isoform often used in the litera-
o describe anaplerotic or ?housekeeping? PEPC
s in leaves of C3 plants (e.g., Lepiniec et al.,
Toh et al., 1994; Rajagopalan et al., 1994) should
ided, because PEPC is not directly involved in
pathway of photosynthesis. In the present paper
ll follow the terminology proposed here.
he context of the question whether PEPC can
as a useful molecular marker in taxonomic and
enetic investigations, in the present study we
nalyzed numerous new partial PEPC sequences.
esults not only increase the number of plants
s that can be compared on the level of PEPC
ces but also include more taxa that are sup-
to mark branching points of plant evolution.
ther, in the present study 70 new PEPC se-
es were analyzed and documented in the EMBL
ank (Table 1). The new sequences represent 24
s of Bryophyta, 1 species of Pteridophyta, and 25
s of Spermatophyta.
previously done (Gehrig et al., 1998a), in the
PEP
Coryneb
Coryneb
Mycoba
Rhodop
Rhodoth
Thermu
Escheri
Haemop
Anabae
Anacyst
Synecho
Chara f
Chara f
Anthoce
Anthoce
Bucegia
Conocep
Fossom
Jungerm
Lunula
Marcha
Preissia
Scapan
Symphy
Batram
Brachyt
Callierg
Dicrane
Dicranu
Funaria
Hypnum
Leptobr
Leucobr
Polytric
Polytric
Rhytidi
Sclerop
Sphagn
Sphagn
Isoetes h
Isoetes d
Lycopod
Selagin
Psilotum
Equiset
Picea ab
Picea ab
Welwits
Sacchar
Sorghum
Sorghum
Sorghum
Triticum
Zea ma
Zea ma
Zea ma
Zea ma
Zea ma
Zea ma
Arabido
Brassic
Brassic
Brassic
264 GEHRIG, HEUTE, AND KLUGETABLE 1
C Partial Sequences Included in the Calculations of the Phylogenetic Trees Shown in Figs. 1?3
Organisms Taxonomic unit Accession No. References
acterium glutamicum 1 Bacteria (a subdivision) X14234 Eikmanns et al. (1989)
acterium glutamicum 2 Bacteria (a subdivision) M25819 Viret and Lemoine (1989)
cterium leprae Bacteria (a subdivision) U00013 Robinson, K. (unpublished)
seudomonas palustris Bacteria (a subdivision) D89668 Inui et al. (1997)
ermus obamensis Bacteria (a subdivision) X99379 Takai et al. (1998)
s sp. Bacteria (a subdivision) D42166 Nakamura et al. (1995)
chia coli Bacteria (g subdivision) X05903 Fujita et al. (1984)
hilus influenzae Bacteria (g subdivision) U00086 Fleischmann et al. (1995)
na variabilis Cyanophyaceae M80541 Luinenburg and Coleman (1992)
is nidulans Cyanophyaceae M11198 Katagiri et al. (1995)
cystis sp. PCC6803 Cyanophyaceae AB001339 Kaneko et al. (1996)
ragilis 1 Charophyceae X95851 Gehrig et al. (1998a)
ragilis 2 Charophyceae X95857 Gehrig et al. (1998a)
ros agrestis Anthocerotae AJ231277 This study
ros punctatus Anthocerotae AJ231278 This study
romanica Hepaticae AJ231280 This study
halum conicum Hepaticae X95853 Gehrig et al. (1998a)
bronia pusilla Hepaticae AJ231304 This study
annia leiantha Hepaticae AJ231287 This study
ria cruciata Hepaticae AJ231289 This study
ntia calcarata Hepaticae AJ231292 This study
quadrata Hepaticae AJ231297 This study
ia nemorea Hepaticae AJ231300 This study
ogyna brongniartii Hepaticae AJ231299 This study
ia pomiformis Musci AJ231279 This study
hecium salebrosum Musci AJ231281 This study
onella cuspidata Musci AJ231282 This study
lla heteromalla Musci AJ231283 This study
m scoparium Musci AJ231284 This study
hygrometrica Musci AJ231285 This study
cupressiforme Musci AJ231286 This study
yum pyriforme Musci AJ231291 This study
yum juniperiodeum Musci AJ231290 This study
hum commune Musci AJ231294 This study
hum formosum Musci AJ231296 This study
adelphus squarrosus Musci AJ231298 This study
odium purum Musci AJ231302 This study
um sp. Musci X95852 Gehrig et al. (1998a)
um palustre Musci AJ231301 This study
istrix Lycopodiatae X95854 Gehrig et al. (1998a)
uriei Lycopodiatae X95859 Gehrig et al. (1998a)
ium annotium Lycopodiatae X95858 Gehrig et al. (1998a)
ella martinii Lycopodiatae AJ252913 This study
nudum Psilotatae X91405 Gehrig et al. (1998a)
um hyemale Equisetatae X95855 Gehrig et al. (1998a)
ies 1 Pinatae X79090 Relle and Wild (1996)
ies 2 Pinatae P51063 Relle and Wild (1996)
chia mirabilis Gnetatae X91404 Gehrig et al. (1998a)
um hybride Poaceae M86661 Henrik et al. (1992)
vulgare 1 Poaceae X59925 Lepiniec et al. (1991)
vulgare 2 Poaceae X65137 Cretin et al. (1990)
vulgare 3 Poaceae X63756 Cretin et al. (1990)
aestivum Poaceae AJ007705 Gonzalez et al. (1998)
ys 1 Poaceae X03613 Izui et al. (1986)
ys 2 Poaceae X15239 Hudspeth and Grula (1989)
ys 3 Poaceae X15238 Hudspeth and Grula (1989)
ys 4 Poaceae X61489 Kawamura et al. (1992)
ys 5 Poaceae AB012228 Dong, L. (unpublished)
ys 6 Poaceae E01120 Katsuki, H. (unpublished)
psis thaliana Brassicaceae AJ131710 Hartung, F. (unpublished)
a juncea 1 Brassicaceae AJ223496 Heiss, S. (unpublished)
a juncea 2 Brassicaceae AJ223497 Heiss, S. (unpublished)
a napus Brassicaceae D13987 Yanai et al. (1994)
Glycine
Glycine
Glycine
Medicag
Pisum s
Vicia fa
Vicia fa
Hydrill
Hydrill
Nicotian
Nicotian
Solanum
Solanum
Flaveria
Flaveria
Flaveria
Flaveria
Flaveria
Drosant
Mesemb
Mesemb
Mesemb
Pereskia
Selenice
Aechme
Neorege
Tilland
Aloe arb
Amaran
Gossypi
Gossypi
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Kalanch
Angraec
Angraec
Chilosc
Dendro
Dendro
Dendro
Dendro
Dendro
Dendro
Dendro
Dendro
Dendro
Dendro
265PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKERTABLE 1?Continued
Organisms Taxonomic unit Accession No. References
max 1 Fabaceae D13998 Tello et al. (1993)
max 2 Fabaceae D10717 Sugimoto et al. (1992)
max 3 Fabaceae AB008540 Hata et al. (1997)
o sativa Fabaceae M83086 Pathirana et al. (1992)
ativum Fabaceae D64037 Suganuma et al. (1997)
ba 1 Fabaceae AJ011302 Golombek, S. (unpublished)
ba 2 Fabaceae AJ011303 Golombek, S. (unpublished)
a verticillata 1 Hydrocharitaceae U65226 Magnin et al. (1996)
a verticillata 2 Hydrocharitaceae U65227 Magnin et al. (1996)
a tabacum 1 Solanaceae X59016 Koizumi et al. (1991)
a tabacum 2 Solanaceae E03014 Yamada, Y. and Sato, F. (unpublished)
tuberosum 1 Solanaceae X67053 Merkelbach et al. (1993)
tuberosum 2 Solanaceae X90982 Panstruga, R. (unpublished)
australasica Asteraceae Z25853 Bauwe, H. (unpublished)
pringlei 1 Asteraceae X64144 Hermans and Westhoff (1992)
pringlei 2 Asteraceae Z48966 Svensson et al. (1997)
trinervia 1 Asteraceae X61304 Poetsch et al. (1991)
trinervia 2 Asteraceae X64143 Hermans and Westhoff (1992)
hemum paxianum Aizoaceae Y17844 This study
ryanthemum crystallinum 1 Aizoaceae X14588 Cushman and Bohnert (1989a)
ryanthemum crystallinum 2 Aizoaceae X14587 Cushman and Bohnert (1989b)
ryanthemum crystallinum 3 Aizoaceae X13660 Rickers et al. (1989)
aculeata Cactaceae X95860 Gehrig et al. (1998a)
reus vitii Cactaceae Y17843 This study
a filicaulis Bromeliaceae AJ252914 This study
lia ampullacea Bromeliaceae X95861 Gehrig et al. (1998a)
sia usneoides Bromeliaceae X91406 Gehrig et al. (1998a)
orescens Asphodelaceae D83052 Honda et al. (1996)
thus hypochondriacus Amarantaceae Z68125 Rydzik and Berry (1996)
um hirsutum 1 Malvaceae AF008939 Vodjani et al. (1997)
um hirsutum 2 Malvaceae AF008940 Vodjani et al. (1997)
oe blossfeldiana 1 Crassulaceae X87818 Gehrig et al. (1995)
oe blossfeldiana 2 Crassulaceae X87819 Gehrig et al. (1995)
oe blossfeldiana 3 Crassulaceae X87820 Gehrig et al. (1995)
oe blossfeldiana 4 Crassulaceae X87821 Gehrig et al. (1995)
oe fedtschenkoi Crassulaceae AJ0010 Menke, H. H. and H. Gehrig (unpublished)
oe gracilipes Crassulaceae AJ231288 This study
oe grandiflora 1 Crassulaceae AJ252918 This study
oe grandiflora 2 Crassulaceae AJ252945 This study
oe x kewensis 1 Crassulaceae AJ252914 This study
oe x kewensis 2 Crassulaceae AJ252915 This study
oe petitiana 1 Crassulaceae AJ231295 This study
oe petitiana 2 Crassulaceae AJ252926 This study
oe pinnata 1 Crassulaceae AJ252919 This study
oe pinnata 2 Crassulaceae AJ252920 This study
oe pinnata 3 Crassulaceae AJ252921 This study
oe pinnata 4 Crassulaceae AJ252922 This study
oe streptantha 1 Crassulaceae AJ252923 This study
oe streptantha 2 Crassulaceae AJ252924 This study
oe streptantha 3 Crassulaceae AJ252925 This study
oe tomentosa 1 Crassulaceae AJ252916 This study
oe tomentosa 2 Crassulaceae AJ252917 This study
um eburneum 1 Orchidaceae X91636 This study
um eburneum 2 Orchidaceae X91631 This study
hista pusilla Orchidaceae X91633 This study
bium crumenatum Orchidaceae AJ252938 This study
bium delicatum Orchidaceae AJ252944 This study
bium farmeri 1 Orchidaceae AJ252939 This study
bium farmeri 2 Orchidaceae AJ252940 This study
bium fimbriatum 1 Orchidaceae AJ252942 This study
bium fimbriatum 2 Orchidaceae AJ252943 This study
bium loddigesie 1 Orchidaceae AJ252933 This study
bium loddigesie 2 Orchidaceae AJ252934 This study
bium mochentum Orchidaceae AJ252941 This study
bium thyrsifolium 1 Orchidaceae AJ252935 This study
presen
1100-b
derive
of the
the ac
conser
relatio
fragm
statist
From
we ob
PEPC
constr
and 2;
plings
bootst
branch
duced
as FIT
result
mente
shown
The
PEPC
gene a
suppo
us
e
yot
ara
res
uen
on
ter
nob
A
es
tst
ta
nch
in
lin
t 1
pr
op
ta
ig
5)
Dendro
Dendro
Microco
Solenan
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Vanilla
Reco
Origin o
Proka
Root
Aeria
Root
Leaf
Cell c
Leaf
Leaf
266 GEHRIG, HEUTE, AND KLUGEt study we compared a distinct PEPC partial
p cDNA sequence and the amino acid sequence
d from it. The sequence is located on the 39 side
coding region of the PEPC gene and comprises
tive center of the enzyme which is sufficiently
vative to reflect larger distances in taxonomic
ns between the species. On the other hand, the
ent was found to be variable enough to allow
ically significant differentiation.
the new and the already published sequences
tained the most detailed phylogenetic trees of
up to now available (Figs. 1?3). The trees were
ucted by neighbor-joining calculations (Figs. 1A
with statistics based on 1000 bootstrap resam-
) and by parsimony analyses (Figs. 1B and 3; 100
rap resamplings). In both types of dendrograms,
es with bootstrap values below 50% were re-
to polytomies. Other distance calculations such
CH and KITSCH were also applied, but since the
s showed the same main topology as that docu-
d in the trees of Figs. 1?3, those data are not
.
comparison of the prokaryotic and eukaryotic
sequences suggests the existence of an ancestral
rising from the g proteobacterial lineage. This
rts the view of R. Kaemmerer (unpublished; cited
in C
mod
kar
sep
rep
infl
dom
bac
cya
lis).
ryot
boo
cero
bra
find
the
por
the
mon
cero
in F
(199
Ant
(He
rick
(200
TABLE 1?Con
Organisms Taxonomic unit
bium thyrsifolium 2 Orchidaceae
bium thyrsifolium 3 Orchidaceae
elia exilis Orchidaceae
gis aphylla Orchidaceae
aphylla 1 Orchidaceae
aphylla 2 Orchidaceae
aphylla 3 Orchidaceae
phalaenopsis 1 Orchidaceae
phalaenopsis 2 Orchidaceae
phalaenopsis 3 Orchidaceae
phalaenopsis 4 Orchidaceae
planifolia 1 Orchidaceae
planifolia 2 Orchidaceae
planifolia 3 Orchidaceae
pompona Orchidaceae
TABLE 2
mmended Nomenclature for the Different Functiona
f the isoform Presumptive function Previous denotatio
ryotic cells Anaplerotic ?
Anaplerotic C3R
l root Anaplerotic C3
nodule Anaplerotic C3
Anaplerotic C3-
ulture Anaplerotic Housekeeping C3
Primary carboxylation C4 photosynthesis
Primary carboxylation CAMhman and Bohnert, 1999). Independent of the
of calculation, the PEPC sequences of the pro-
es form a distinct cluster (Fig. 1) with three
ted branches (bootstrap support 93?100%)
enting g proteobacteria (E. coli, Haemophilus
za), a proteobacteria (Thermus sp., Rhodopseu-
as palustris, Rhodothermus obamensis, Myco-
ium leprae, Corynebacterium glutamicum), and
acteria (Anacystis nidulans, Anabaena variabi-
t the base of the cluster representing the euca-
, there is a small branch separating with high
rap support the two species of hornworts (Antho-
e: Anthoceros punctatus, A. agrestis) from the
comprising Chara and all the land plants. The
g that Anthoceros branches off before Chara from
eage leading to the land plants (bootstrap sup-
00%) was unexpected because it is in contrast to
esent widely held view that the land plants are
hyletic (e.g., Qui and Palmer, 1999). The Antho-
e form a separate cluster in the trees presented
1. This result is contrary to those of Capesius
and Bopp and Capesius (1996), which show the
cerotae located among the Jungermanniidae
ticae), and to those of Mishler et al. (1994), Ken-
nd Crane (1997a,b), and Graham and Wilcox
, which place the hornworts between the liver-
ed
Accession No. References
AJ252936 This study
AJ252937 This study
X91635 This study
X91632 This study
X91634 This study
AJ252927 This study
AJ252928 This study
AJ252948 This study
AJ252930 This study
AJ252931 This study
AJ252932 This study
X87148 Gehrig et al. (1998b)
X87149 Gehrig et al. (1998b)
AJ249988/9 This study
AJ252929 This study
EPC Isoforms: Previous and New Denotations
Denotation after Toh et al. (1994) New denotation
Bacterial ppc-aP
C3-1 ppc-aR
? ppc-aR
C3-2 ppc-aR
C3-3 ppc-aL
? ppc-aL
C4 ppc-C4ho
pa
a
1)
l P
ntinuCAM ppc-CAM
worts
reprod
phyte
worts,
the ho
phyte
trache
can be
strap v
with t
Antho
tion, A
PEPC
remai
suppo
FIG.
gene. T
the nod
in detai
box see
267PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKERand the mosses. On the other hand, studies of
uctive and structural innovations in the gameto-
and sporophyte generations of hornworts, liver-
and mosses (Renzagali et al., 2001) suggest that
rnworts represent the earliest divergent embryo-
clade, with the moss/liverwort clade as sister to
ophytes. From our present results, hornworts
regarded a basal group separated by high boot-
alues (100%). Our results are in good agreement
hose of Waters et al. (1992), which show the
cerotae as a sister clade to the Musci. In addi-
nthocerotae show an amino acid composition of
that is completely different from that of the
ning species investigated. Moreover, our results
wor
land
O
form
In t
com
seq
the
obta
Sin
plan
PEP
of t
con
1. (A) Neighbor-joining phylogenetic tree of 143 amino acid sequen
he tree comprises all PEPC sequences up to now known; among the
es indicate bootstrap values (values less than 50% are not shown). T
l in Fig. 2. (B) Parsimony phylogenetic tree of the same PEPC amino
Fig. 3).rt the opinion by Sluiman (1985) that the horn- phycearepresent an entirely independent derivation of
lants.
results fit with the view that the charophytes
paraphyletic group relative to the land plants.
s context it would have been interesting also to
re other algae on the level of the PEPC partial
ce. However, with the PEPC primer pair used in
esent studies, PCR amplification products were
ed only with Chara and not with other algae.
our primers were derived from PEPC of higher
this finding can be taken as a hint that the
structure of Chara is related much closer to that
land plants than to that of the algae. This is
tent with the phylogenetic position of the Charo-
based on an approx 1100-bp fragment (39 side) of the PEPC
70 were first analyzed in the present study. Numbers above
ox comprising the sequences of the Spermatophyta is shown
id sequences as shown in A (for details of the Spermatophytats
p
ur
a
hi
pa
uen
pr
in
ce
ts
C
he
sis
ces
m,
he b
ace in relation to the land plants and algae as
FIG. 2. Details of the Spermatophyta box of the neighbor-joining tree shown in Fig. 1A.
FIG. 3. Details of the Spermatophyta box of the parsimony tree shown in Fig. 1B.
propos
analyz
new p
Exc
(Figs.
bryoph
strap
ing th
gests t
in par
Capes
the am
forms
Hepat
to the
58%).
Antho
and M
The
invest
matop
(boots
provid
contra
pterid
dent o
grams
specie
separa
karyot
tively)
repres
gymno
donae
the la
quenc
though
partic
quenc
Welwi
bootst
Classi
group
molecu
more c
sperm
al., 20
shown
favor
With
the PE
but al
to the
might
eratio
analys
tween
ai
se
sen
he
he
to
an
sh
rly
In
t
s
to
Or
cie
uen
. 4
ve
s S
an
ary
ary
ph
ido
ma
270 GEHRIG, HEUTE, AND KLUGEed by Qui and Palmer (1999). To be able to
e PEPC sequences in algae, we need to construct
rimers.
ept for the hornworts, in the phylogenetic trees
1A and 1B) the PEPC sequences of the other 24
ytes investigated so far form two clusters (boot-
support 88 and 100%), with one branch compris-
e Hepaticeae and the other the Musci. This sug-
hat these two groups of Bryophyta have evolved
allel (Waters et al., 1992; Mishler et al., 1994;
ius and Stech, 1997). It is worth mentioning that
ino acid sequences of the Anthoceros PEPC iso-
show a significantly lower homology to the other
iceae and Musci (40 and 47%, respectively) than
two PEPC isoforms of Chara fragilis (51 and
This is further support of the view that the
cerotae have evolved separately from Hepaticae
usci (Schuster, 1984).
PEPC sequences of the 6 species of Pteridophyta
igated up to now form a sister group to the Sper-
hyta, with three parallel polytomic branches
trap values 49, 55, and 94%). The present data
e further evidence in favor of the view that, in
st to former assumptions, the mosses but not the
ophytes represent the first land plants. Indepen-
f the mode of calculation, in the PEPC dendro-
shown in Figs. 1A and 1B, the Spermatophyta
s form one large common cluster which is clearly
ted from that of the archegoniates and pro-
es (bootstrap support by 94 and 82%, respec-
. The PEPC sequences of the Spermatophyta
ent 53 plant species in 16 plant families (2
sperms, 51 angiosperms, with 8 of the Dicotyle-
and 6 of the Monocotyledonae; Table 3). Within
rge spermatophytean cluster the PEPC se-
es form different branches (Figs. 2 and 3), al-
some of them have low bootstrap support. A
ularly interesting finding concerns the PEPC se-
es of the conifer Picea abies and the gnetophyt
tschia mirabilis (Fig. 2) showing that with high
rap support these two species cluster together.
cally the gnetophytes are considered the sister
of the angiosperms. However, there are now
lar data which imply that the gnetophytes are
losely related to the conifers than to the angio-
s (Winter et al., 1999; Bowe et al., 2000; Chaw et
00; Donoghue and Doyle, 2000). The PEPC tree
in Fig. 2 provides further strong support in
of the latter view.
in the cluster representing the Spermatophyta
PC sequences not only arrange according to taxa
so within a taxon apparently arrange according
ir assumed specific function. This phenomenon
reflect functional diversification during the gen-
n of paralogous PEPC genes. However, a detailed
is of the presumably complex relationships be-
rem
cau
pre
in t
T
ing
inst
not
clea
4).
into
form
ing
the
spe
seq
(Fig
O
Thi
Org
Prok
Euk
Bryo
Pter
Sperparalogous and orthologous PEPC genes has to with tn beyond the scope of the present treatise, be-
it would require much more information than at
t is available on the existence of PEPC isoforms
single plant species.
mentioned clustering of PEPC sequences accord-
the assumed specific function can be observed for
ce in the genera Flaveria and Kalanchoe (data
own in detail), but can be seen particularly
in the case of the Poaceae and Orchidaceae (Fig.
the Poaceae the PEPC sequences are separated
hree branches representing the functional iso-
ppc-C4, ppc-aL, and ppc-aR (denotation accord-
the nomenclature outlined in Table 2). Also in
chidaceae (28 PEPC sequences representing 15
s in 6 genera) there is evidence that the PEPC
ces branch according to their proposed functions
). In the orchids we found two major clusters,
TABLE 3
rview of the 143 PEPC Isoforms Considered in
tudy and the Taxonomic Position of the Donor
ism
Taxonomic unit
Number of
considered
species
Numbers of
PEPC sequences
analyzed in
the considered
species
ota
a Subdivision 6 6
g Subdivision 2 2
Cyanophyceae 3 3
ota
Charophyceae 1 2
yta Anthocerotae 2 2
Marchantiatae 9 9
Bryatae 15 15
phyta Psilotatae 1 1
Lycopodiatae 4 4
Equisetatae 1 1
tophyta Gymnospermae
Pinatae 1 2
Gnetatae 1 1
Angiospermae
Dicotyledoneae
Malvaceae 1 2
Crassulaceae 9 21
Solanaceae 2 4
Aizoaceae 2 4
Asteraceae 3 5
Fabaceae 4 7
Brassicacae 3 4
Cactaceae 2 2
Monocotyledoneae
Poaceae 4 11
Bromeliaceae 3 3
Orchidaceae 15 28
Amarantaceae 1 1
Asphodelaceae 1 1
Hydrocharitaceae 1 2he smaller cluster comprising all the isoforms
that w
roots
presse
which
plant
PEPC
FIG.
of the c
function
the conc
materia
roots. Q
271PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKERe have identified in nonphotosynthetic aerial
and the larger cluster comprising isoforms ex-
d in photosynthetic organs. In those cases in
we have shown CAM performance when the
tissue was extracted, we assumed that the found
nill
root
the
ing
lim
4. Interrelationships between the position of PEPC sequences in th
oncerned PEPC isoforms, exemplified for the Poaceae and Orchida
follows the suggestions published in the literature (see Table 1). Fo
erned isoform was the only or the mainly expressed isoform in plant
l was extracted (data not shown). The denotation ppc-aR refers to th
uestion marks indicate unknown function of the concerned PEPC iisoform was CAM related. For instance, in Va- thesislanifolia, the ?isoform 2? expressed in the aerial
ppc-aR) appears in a cluster other than that of
oform 1? cluster expressed in the CAM-perform-
ves. Since in the case of Dendrobium, because of
tion in the plant material the mode of photosyn-
eighbor-joining tree (Fig. 2) and the likely specific functions
e. In the Poaceae the attribution of the isoforms to a given
he Orchidaceae, attribution to CAM is based on the fact that
terial for which we have shown CAM performance when the
ct that the isoform was found in non-CAM-performing aerial
rm.a p
s (
?is
lea
ita
e n
cea
r t
ma
e fa
sofocould not be investigated, in this genus the def-
inite a
open.
resent
all spe
CAM
PEPC
be sho
type.
Amo
specie
lenang
consis
aerial
being
forms
the P
roots o
cluste
in CAM
non su
use of
cataly
they o
isoform
The
findin
phytes
relate
levels
the ev
man a
Alto
that
marke
future
plants
the ev
PEPC
the co
may b
full-le
and fi
the va
quenc
We th
and Fa
Monika
acknow
Forschu
tion.
Andreo,
plant
FEBS
er,
T r
, M
y p
e, L
ed
mn
e co
s,
mat
siu
e n
.
siu
osse
w, S
D.
ono
nife
gw
979
rich
let,
te c
nu
in,
990
te c
: 65
in,
da
in
pre
ma
e P
esem
44.
ma
e g
yan
ma
n o
ol.
ma
assu
ma
boli
ol.
ma
hn
o i
ean
ll 1
ogh
mi
sle
on
tle
rde
an
989
ium
272 GEHRIG, HEUTE, AND KLUGEttribution of PEPC isoforms to CAM remains
However, because the Dendrobium species rep-
succulent-leaf epiphytes, and to our knowledge
cies of that genus thereupon investigated are
plants, we believe that most of the concerned
isoforms labeled in Fig. 4 by a question mark can
wn by future work to belong to the ppc-CAM
ng the investigated orchids there were three
s (Microcoelia exilis, Chilochista pusilla, So-
is aphylla) in which the photosynthetic organs
t of chloroplasts containing CAM-performing
roots (Winter, 1985), with leaves and shoot axes
largely reduced. As Fig. 4 shows, the PEPC iso-
of these ?shootless? orchids do not cluster with
EPC isoforms of the nonphotosynthetic aerial
f the Vanilla and Dendrobium species but rather
r with the PEPC isoforms found, in other species,
-performing leaves. This interesting phenome-
ggests that the shootless orchids do not make
the root-inherent ppc-aR isoform of PEPC to
ze the initial b carboxylation in CAM. Rather,
bviously express for this function an additional
presumably specifically related to CAM.
results of our present study support the previous
gs by Gehrig et al. (1998a) that in the spermato-
the PEPC isoforms assumed to be functionally
d to CAM are widely dispersed over the different
of taxa. This is in harmony with the view that
olution of CAM is of polyphyletic origin (Cush-
nd Bohnert, 1997, 1999).
gether, the present study strengthens the view
PEPC sequences provide valuable molecular
rs which may help to answer open questions in
phylogenetic studies of microorganisms and
. They can also contribute to better knowledge of
olution of metabolic pathways in which the
is involved. Our study has also shown that, in
ntext of molecular phylogeny and taxonomy, it
e sufficient to compare suitable partial instead of
ngth PEPC sequences. This helps to save time
nancial resources, thus considerably increasing
lue of PEPC nucleotide and amino acid se-
es as widely applicable molecular markers.
ACKNOWLEDGMENTS
ank Catharine von Glassow, Ute Reck, Kerstin Weitmann,
nka Rosic for practical assistance. The careful work by
Medina-Espan?a in preparing the manuscript is gratefully
ledged. The investigations were supported by the Deutsche
ngsgemeinschaft and by a grant from the FAZIT Founda-
REFERENCES
C. S., Gonzales, D. H., and Iglesias, A. A. (1987). Higher
Bogl
IS
Bopp
om
Bow
se
gy
ar
Brun
te
Cape
th
63
Cape
m
Cha
J.
M
co
Chir
(1
en
Chol
va
An
Cre?t
(1
va
18
Cre?t
Ga
ily
ex
Cush
th
M
67
Cush
th
br
Cush
tio
Ec
Cush
cr
Cush
ta
Bi
Cush
Bo
tw
lac
Ce
Don
De
Dres
?M
Cu
Ga
Eikm
(1
terphosphoenolpyruvate carboxylase: Structure and regulation.
Lett. 213: 1?8.
Felsens
sion 3D., and Simpson, B. (1996). Phylogeny of Agavaceae based on
DNA sequence variation. Oecologia 83: 1225?1235.
., and Capesius, I. (1996). New aspects of Bryophyte taxon-
rovided by a molecular approach. Bot. Acta 109: 368?372.
. M., Coat, G., and dePamphilis, C. W. (2000). Phylogeny of
plants based on all three genomic compartments: Extant
osperms are monophylethic and Gnetales closest relatives
nifers. Proc. Natl. Acad. Sci. USA 97: 4092?4097.
T., White, T., and Taylor, J. (1991). Fungal molecular sys-
ics. Annu. Rev. Ecol. Syst. 22: 525?564.
s, I. (1995). A molecular phylogeny of bryophytes based on
uclear encoded 18S rRNA genes. J. Plant Physiol. 146: 59?
s, I., and Stech, M. (1997). Molecular relationships within
s based on 18S rRNA gene sequences. Nova Hed. 64: 3?4.
., Parkinson, C. L., Cheng, Y., Vincent, T. M., and Palmer,
(2000). Seed plant phylogeny from all three plant genomes:
phyly of extant gymnospoerms and origin of Gnetales from
rs. Proc. Natl. Acad. Sci. USA 97: 4086?4091.
in, J. M., Przybyla, A. E., McDonald, R. J., and Rutter, W. J.
). Isolation of biologically active ribonucleic acid from sources
ed in ribunucleases. Biochemistry 18: 5294?5299.
R., Vidal, J., and O?Leary, M. H. (1996). Phosphoenolpyru-
arboxylase: A ubiquitous, highly regulated enzyme in plants.
. Rev. Plant Physiol. Plant Mol. Biol. 47: 273?298.
C., Keryrer, E., Tagu, D., Lepiniec, L., Vidal, J., and Gadal, P.
). Complete cDNA sequence of Sorghum phosphoenolpyru-
arboxylase involved in C4 photosynthesis. Nucleic Acid Res.
8.
C., Santi, S., Keryrer, E., Lepiniec, L., Tagu, D., Vidal, J., and
l, P. (1991). The phosphoenolpyruvate carboxylase gene fam-
Sorghum: Promoter structure, amino acid sequences and
ssion of genes. Gene 99: 87?94.
n, J. C., and Bohnert, H. J. (1989a). Nucleotide sequence of
pc2 gene encoding a housekeeping isoform of PEPC from
bryanthemum crystallinum. Nucleic Acid Res. 17: 6743?
n, J. C., and Bohnert, H. J. (1989b). Nucleotide sequence of
ene encoding a CAM specific isoform of PEPC from Mesem-
themum crystallinum. Nucleic Acid Res. 17: 6745?6746.
n, J. C., and Bohnert, H. J. (1996). Transcriptional activa-
f CAM genes during development and environmental stress.
Stud. 114: 135?158.
n, J. C., and Bohnert, H. J. (1997). Molecular genetics of
lacean acid metabolism. Plant Physiol. 113: 667?676.
n, J. C., and Bohnert, H. J. (1999). Crassulacean acid me-
sm: Molecular genetics. Annu. Rev. Plant Physiol. Plant Mol.
50: 305?332.
n, J. C., Meyer, G., Michalowski, C. B., Schmitt, J. M., and
ert, H. J. (1989). Salt stress leads to differential expression of
sogenes of phosphoenolpyruvate carboxylase during crassu-
acid metabolism induction in the common ice plant. Plant
: 715?725.
ue, M. J., and Doyle, J. A. (2000). Seed plant phylogeny:
se of the anthophyte hypothesis? Curr. Biol. 10: R106?R109.
r, R. L., and Chase, M. W. (1995). Whence the orchids? In
ocotyledons: Systematics and Evolution? (P. J. Rudall, D. F.
r, and C. J. F. Humphries, Eds.), pp. 217?226. Royal Botanic
ns, Kew.
ns, B. J., Folletie, M. T., Griot, M. U., and Sinskey, A. J.
). The phosphoenolpyruvate carboxylase gene of Corynebac-
glutamicum. Mol. Gen. Genet. 218: 330?339.
tein, J. (1993). PHYLIP (Phylogeny Inference Package) ver-
.5c. Univ. of Washington.
Fleischm
Smith
semb
Fujita,
mary
J. Bio
Gehrig,
cation
sulac
Poelln
Gehrig,
know
boxyl
46: 10
Gehrig,
phosp
of the
A phy
1223.
Gonzale
F. J.
carbo
Physi
Graham
of gen
trans
Hata, S
nodul
shows
form.
Henrik,
expre
phoen
Hepper
Herman
isofor
Speci
Honda,
for a
plant
Cell P
Hudspe
the m
isozym
589.
Inui, M
(1997
vate c
dopse
Izui, K.
sada,
quenc
synth
Kaneko
mura
Kimu
Naru
Wata
Seque
rium
entire
DNA
Katagir
Nucle
of cya
am
d I
rbo
NA
volv
rick
pla
rick
ca
tion
ura
mb
e, M
is o
um
aly
ltur
ko,
osp
5?8
nie
etin
e so
ol.
nie
mp
osp
ant
nie
993
um
ol.
nie
osp
tion
enb
tio
ruv
icro
nin
osp
t wi
er,
oen
.
kelb
ler,
NA
ant
ler
rba
ap
ae
am
oni
te
Bio
ira
falf
tio
lle
sch
273PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKERann, R. D., Adams, M. D., White, O., Clayton, R. A., and
, H. O. (1995). Whole genome random sequencing and as-
ly of Haemophilus influenzae rd. Science 269: 496?512.
N., Miwa, T., Ishijima, K., and Katsuki, H. (1984). The pri-
structure of phosphoenolpyruvate carboxylase of E. coli.
chem. 95: 909?916.
H. H., Taybi, T., Kluge M., and Brulfert, J. (1995). Identifi-
of multiple PEPC isogenes in leaves of the facultative cras-
ean acid metabolism (CAM) plant Kalanchoe blossfeldiana
. cv. Tom Thumb. FEBS Lett. 377: 399?402.
H. H., Heute, V., and Kluge M. (1998a). Towards a better
ledge of the molecular evolution of phosphoenolpyruvate car-
ase by comparison of partial cDNA sequences. J. Mol. Evol.
7?114.
H. H., Faist, K., and Kluge, M. (1998b). Identification of
hoenolpyruvate carboxylase isoforms in leaf, stem, and roots
obligate CAM plant Vanilla planifolia Salib. (Orchidaceae):
siological and molecular approach. Plant Mol. Biol. 38: 1215?
z, M. C., Osuna, L., Echevarria, C., Vidal, J., and Cejudo,
(1998). Expression and localization of phosphoenolpyruvate
xylase in developing and germinating wheat grains. Plant
ol. 116: 1249?1258.
, L. K. E., and Wilcox, L. W. (2001). The origin of alternation
erations in land plants: A focus on matrophy and hexose
porter. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 29: 757?766.
., Izui, K., and Kouchi, H. (1997). Expression of a soybean
e-enhanced phosphoenolpyruvate carboxylase gene that
striking similarity to another gene for a housekeeping iso-
Plant J. 7: 198?201.
A. H., Martin, T., and Sun, S. S. M. (1992). Structure and
ssion of a sugarcane gene encoding a housekeeping phos-
olpyruvate carboxylase. Plant Mol. Biol. 20: 663?671.
le, D. (1997). Alignment editor, version 3.5a. Heidelberg.
s, J., and Westhoff, P. (1992). Homologous genes for the C4
m of phosphoenolpyruvate carboxylase in C3 and C4 Flaveria
es. Mol. Gen. Genet. 234: 275?284.
H., Okamoto, T., and Shimada, H. (1996). Isolation of a cDNA
phosphoenolpyruvate carboxylase from a monocot CAM-
, Aloe arborescens: Structure and its gene expression. Plant
hysiol. 37: 881?888.
th, R. L., and Grula, J. W. (1989). Structure and expression of
aize gene encoding the phosphoenolpyruvate carboxylase
e involved in C4 photosynthesis. Plant Mol. Biol. 12: 579?
., Dumay, V., Zahn, K., Yamagata, H., and Yukawa, H.
). Structural and functional analysis of the phosphoenolpyru-
arboxylase gene from the purple nonsulfur bacterium Rho-
udomonas palustris No. 7. J. Bacteriol. 179: 4942?4945.
, Ishijima, S., Yamaguchi, Y., Katagiri, F., Murata, T., Shige-
K., Sugiyama, T., and Katsuki, H. (1986). Cloning and se-
e analysis of cDNA encoding active PEPC of the C4 photo-
esis in maize. Nuc. Acid Res. 14: 1615?1628.
, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Naka-
, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S.,
ra, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N.,
o, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T.,
nabe, A., Yamada, M., Yasuda, M., and Tabata, S. (1996).
nce analysis of the genome of the unicellular cyanobacte-
Synechocystis sp. PCC6803: Sequence determination of the
genome and assignment of potential protein-coding regions.
Res. 3: 109?136.
i, F., Kodaki, T., Fuijta, N., Izui, K., and Katsuki, H. (1985).
Kaw
an
ca
cD
in
Ken
of
Ken
sifi
tu
Kim
Ca
Klug
ys
Koiz
an
cu
Latz
ph
80
Lepi
Cr
th
M
Lepi
Co
ph
Pl
Lepi
(1
gh
Bi
Lepi
Ph
lu
Luin
iza
py
M
Mag
ph
co
Melz
ph
60
Mer
ta
cD
pl
Mish
Ga
Ch
alg
Nak
Cl
va
J.
Path
Al
iza
tro
Poet
otide sequence of the phosphoenolpyruvate carboxylase gene
nobacterium Anacystis nidulans. Gene 38: 265?269.
of ph
nerviaura, T., Shigesada, K., Toh, H., Okumura, S., Yanagisawa, S.,
zui, K. (1992). Molecular evolution of phosphoenolpyruvate
xylase for C4 photosynthesis in maize: Comparison of its
sequence with a newly isolated cDNA encoding an isozyme
ed in the anaplerotic function. J. Biochem. 112: 147?154.
, P., and Crane, P. R. (1997a). The origin and early evolution
nts on land. Nature 389: 33?39.
, P., and Crane, P. R. (1997b). ?The Origin and Early Diver-
tion of Land Plants: A Cladistic Study.? Smithsonian Insti-
Press, Washington, DC.
, M. (1983). ?The Neutral Theory of Molecular Evolution.?
ridge Univ. Press, Cambridge, UK.
., and Ting, I. (1978). Crassulacean acid metabolism: Anal-
f an ecological adaptation. Ecol. Stud. 30.
i, N., Sato, F., Terano, Y., and Yamada, Y. (1991). Sequence
sis of cDNA encoding phosphoenolpyruvate carboxylase from
ed tobacco cells. Plant Mol. Biol. 17: 535?539.
E., and Kelly, G. J. (1983). The many-faceted function of
hoenolpyruvate carboxylase in C3 plants. Physiol. Veg. 21:
15.
c, L., Santi, S., Keryer, E., Amiet, V., Vidal, J., Gadal, P., and
, C. (1991). Complete nucleotide sequence of one member of
rghum phosphoenolpyruvate carboxylase gene family. Plant
Biol. 17: 1077?1079.
c, L., Keryer, E., Tagu, D., Gadal, P., and Cretin, C. (1992).
lete nucleotide sequence of a sorghum gene coding for the
hoenolpyruvate carboxylase involved in C4 photosynthesis.
Mol. Biol. 19: 339?342.
c, L., Keryer, E., Philippe, H., Gadal, P., and Cretin, C.
). The phosphoenolpyruvate carboxylase gene family of sor-
: Structure, function and molecular evolution. Plant Mol.
21: 487?502.
c, L., Vidal, J., Chollet, R., Gadal, P., and Cretin, C. (1994).
hoenolpyruvate carboxylase: Structure, regulation and evo-
. Plant Sci. 99: 111?124.
urg, I., and Coleman, J. R. (1992). Identification, character-
n and sequence analysis of the gene encoding phosphoenol-
ate carboxylase in Anabaena variabilis PCC7120. J. Gen.
biol. 138: 685?692.
, N., Reiskind, J. B., and Bowes, G. (1996). Identification of
hoenolpyruvate carboxylase isoforms from an aquatic mono-
th inducible C4-type photosynthesis. Plant Physiol. 8: 72?72.
E., and O?Leary, M. (1987). Anaplerotic fixation by phos-
olpyruvate carboxylase in C3 plants. Plant Physiol. 84: 58?
ach, S., Gehlen, J., Denecke, M., Hirsch, H. J., and Kreuz-
F. (1993). Cloning, sequence analysis and expression of a
encoding active phosphoenolpyruvate carboxylase of the C3
Solanum tuberosum. Plant Mol. Biol. 23: 881?888.
, B. D., Lewis, L. A., Buchheim, M. A., Renzaglia, K. S.,
ry, D. J., Delwiche, C. F., Zechman, F. W., Kantz, T. S., and
man, R. L. (1994). Phylogenetic relationships of the green
and bryophytes. Ann. Missiouri Bot. Gard. 81: 451?483.
ura, T., Yoshioka, I., Takahashi, M., and Izui, K. (1995).
ng and sequence analysis of the gene for phosphoenolpyru-
carboxylase from an extreme thermophile, Thermus sp.
chem. 118: 319?324.
na, S. M., Vance, C. P., Miller, S. S., and Gantt, J. S. (1992).
a root nodule phosphoenolpyruvate carboxylase: Character-
n of the cDNA and expression in effective and plant-con-
d ineffective nodules. Plant Mol. Biol. 20: 437?450.
, W., Hermans, J., and Westhoff, P. (1991). Multiple cDNAs
osphoenolpyruvate carboxylase in the C4 dicot Flaveria tri-
. FEBS Lett. 292: 133?136.
Qiu, Y. L., and Palmer, J. D. (1999). Phylogeny of early land plants:
Insights from genes and genomes. Trends Plant Sci. 4: 26?30.
Rajagopalan, A. V., Tirumala, D. M., and Raghavendra, A. S. (1994).
Molecular biology of C4 phosphoenolpyruvate carboxylase struc-
ture, regulation and genetic engineering (Review). Photosynth.
Res. 39: 115?135.
Relle, M., and Wild, A. (1996). Molecular characterization of a phos-
phoenolpyruvate carboxylase in the gymnosperm Picea abies (Nor-
way spruce). J. Plant Physiol. 149: 225?228.
Renzagali, K. S., Duff, R. J., Nickrent, D. L., and Garbary, D. J.
(2001). Vegetative and reproductive innovations of early land
plants: Implications for a unified phylogeny. Philos. Trans. R. Soc.
Lond. B. Biol. Sci. 29: 769?793.
Rickers, J., Cushman, J., Michalowski, C., Schmitt, J., and Bohnert,
H. J. (1989). Expression of the CAM-form of phosphoenolpyruvate
carboxylase and nucleotide sequence of a full length cDNA from
Mesembryanthemum crystallinum. Mol. Gen. Genet. 215: 447?454.
Rydzik, E., and Berry, J. (1996). The C4 photosynthetic phosphoenol-
pyruvate carboxylase from grain amaranth. Plant Physiol. 110:
713?715.
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:
406?425.
Schulle
regul
vate c
Schuste
Ed.),
Sluiman
green
Suganu
cDNA
pea a
tive p
Sugimo
and K
phoen
20: 74
Svensso
the en
ylase: A comparison of the orthologous PPCA phosphoenolpyru-
vate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei
(C3). Eur. J. Biochem. 246: 452?460.
Takai, K., Sako, Y., and Uchilda, A. (1998). The gene for phos-
phoenolpyruvate carboxylase from an extremely thermophilic bac-
terium, Rhodothermus obamensis: Cloning, sequencing and over
expression in Escherichia coli. Microbiology 144: 1423?1434.
Tello, A. V., Whittier, R. F., Kawasaki, T., Sugimoto, T., Kawamura,
Y., and Shibata, D. (1993). Sequence of a Soybean (Glycine max L.)
phosphoenolpyruvate carboxylase cDNA. Plant Physiol. 103:
1025?1026.
Toh, H., Kawamura, T., and Izui, K. (1994). Molecular evolution of
phosphoenolpyruvate carboxylase. Plant Cell Environ. 17: 31?43.
Utter, M. F., and Kohlenbrander, H. M. (1972). Formation of oxala-
cetate by CO2-fixation on phosphoenolpyruvate. In ?The Enzyme?
(Boyer, Ed.), Vol. 4, pp. 117?170. Academic Press, New York.
Viret, J. F., and Lemoine, Y. (1989). Cloning and nucleotide sequence
of the phosphoenolpyruvate carboxylase-coding gene of Corynebac-
terium glutamicum ATCC13032. Gene 77: 237?251.
Vodjani, F., Kim, W., and Wilkins, T. A. (1997). Phosphoenolpyru-
vate carboxylase cDNA from developing cotton (Gossypium hirsu-
tum) fibers. Plant Physiol. 115: 315?315.
Waters, D. A., Buchheim, M. A., Dewery, R. A., and Chapman, R. L.
(1992). Preliminary inferences of the phylogeny of bryophytes from
cle
6.
ter,
ech
. 32
ter,
eis
ore
tl.
i, Y
a n
ns
otec
awa
lor
cea
d r
274 GEHRIG, HEUTE, AND KLUGEr, K. A., Turpin, T. H., and Plaxton, W. C. (1990). Metabolite
ation of partially purified soybean nodule phosphoenolpyru-
arboxylase. Plant Physiol. 94: 1429?1435.
r, R. M. (1984). In ?New Manual of Bryology? (R. Schuster,
Vol. 2, p. 1071. Hattori Bot. Lab., Nichinan, Japan.
, H. J. (1985). A cladistic evaluation of the lower and higher
plants. Plant Syst. Evol. 149: 217?232.
ma, N., Okada, Y., and Kanayama, Y. (1997). Isolation of a
for nodule-enhanced phosphoenolpyruvate carboxylase from
nd its expression in effective and plant-determined ineffec-
ea nodules. J. Exp. Bot. 48: 1165?1173.
to, T., Kawasaki, T., Kato, T., Whittier, R. F., Shibata, D.,
awamura, Y. (1992). cDNA sequence and expression of phos-
olpyruvate carboxylase gene from soybean. Plant Mol. Biol.
3?747.
n, P., Blaesing, O. E., and Westhoff, P. (1997). Evolution of
zymatic characteristics of C4 phosphoenolpyruvate carbox-
nu
46
Win
M
pp
Win
Th
m
Na
Yana
sic
tro
Bi
Yuk
Ch
da
anar-encoded ribosomal RNA sequences. Am. J. Bot. 79: 459?
K. (1985). Crassulacean acid metabolism. In ?Photosynthetic
anisms and Environment? (J. Barber, and N. R. Baker, Eds.),
9?387. Elsevier, Amsterdam.
K. U., Becker, A., Muenster, T., Kim, J. T., Saedler, H., and
sen, G. (1999). MADS-box genes reveal that gnetophytes are
closley related to conifers than to flowering plants. Proc.
Acad. Sci. USA 96: 7342?7347.
., Okumura, S., and Shimada, H. (1994). Structure of Bras-
apus phosphoenolpyruvate carboxylase genes: Missing in-
causing polymorphisms among gene family members. Biosci.
hnol. Biochem. 58: 950?953.
, T., Ohba, H., Cameron, K. M., and Chase, M. W. (1996).
oplast DNA phylogeny of subtribe Dendrobiinae (Orchi-
e): Insights from combined analysis based on rbcL sequences
estriction site variation. J. Plant Res. 109: 169?176.