Indi an Journal of Experimental Biology
Vol. 39, Jul y 2001 , pp. 643-649
Immunological characteristics of PEP carboxylase from leaves of Cr, C4- and
CrC 4 intermediate species of Alternanthera - Comparison with selected
Cr and C4- plants
J Gayathri, K Parvathi , Bhaskarrao Chinthapalli, Peter Westhoff* & AS
Ra ghavendra*'~
Department of Plant Sciences. School of Life Sciences, University of Hyderabad,
Hyderabad 500 046. India
*Universitat DUsseldorf, lnstitut fUr Entwicklun gs- und Molekularbiolog ie der Pnanzen,
Universitatstrasse I. D-40225 DU sseldorf. Germany
Received 14 August 2000; revised 12 March 2001
Immunological cross-reacti vity of phosphoenolpyruvate carboxy lase (PEPC) in leaf ex tracts of C 3-, C4- and C3-C4
intermediate spec ies of Alternanthera (along with a few other C3 - and C4 - plants) was studied using anti-PEPC antibodi es
rai sed again st PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Altemanthera, namely
Amaranthac e:~e) . Antibodies were also rai sed in rabbits against the purified PEPC from Zea mays (C 4 - monocot - Poaceae)
as well as Al!emanthera p 1111p,ens (C 4 - dicot - Amaranthaceae). Monospecificity of PEPC-anti serum was confirmed by
immunoprec ipitati on. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial
immunodiffusion. Cros- reac ti vit y of PEPC in leaf extracts from se lected C3. , C4., and CTC4 intermediate species (including th ose of Altemanthera) was exam ined using Ouchterl ony doub le diffusion and Western bl ots. Anti-PEPC anti serum
rai sed against A. lnpochondriacus enzy me showed high cross-reactivi ty with PEPC in leaf extracts of A. hvpochondriacus
or Amaranthus viridis or Alternanth era pungens (all C4 dicots). but limited cross-reactivi ty wit h that of Zea mays, Sorghum
or PenniseTUIII (all C4 monocots) . Interestin gly, PEPC in leaf extracts of AITemalllhera tenet/a. A. Jicoides . Panh enium
hysteroplwrus (CrC 4 intermedi ates) ex hibited stronger cross-reactivity (with anti-serum rai sed against PEPC from
Amaranthus hypochondriacus) than that of Pisum smivum. Co11unelina bengha/ensis, AITenanthera sessilis (C 3 plants).
Further studies on cross-reactivities of PEPC in leaf ex tracts of these plants with anti-PEPC anti sera rai sed against PEPC
fro m leaves of Zea mays or Altemanthcra pungens confirmed two points - (i) PEPC of CTC4 intermed iate is di stinct from
C3 species and intermediate between th ose of C3- and C4-spec ies; and (ii ) PEPC of C4-dicots was closer to that of Crspec ies
or C_rC 4 intermediates (di cots) than to that of C4-monocots.
Being a key enzyme, pro pe rti es of PEPC can be used
as an interesting tool to assess the evo luti o nary re lati o nships between C 3 and C 4 plants and C 3-C4 intermediates 1·3 • Our studi es o n biochemical properties of
th e enzyme demo nstrated that PEPC from C 3-C4 intermediates of A/ternan th era (A . tenet/a and ficoides)
exhibits an inte rmediate statu s betwee n those of
C 3- (A . sessi/is) and C 4 -spec ies (A. pungenst
A rapid conventional me thod for purification of
PEPC from th e leaves of A111aranthus hyp ochondriacus (another C 4 -dicot and belonging to the same famil y of A/temanthe ra, name ly Amaranthaceae) has
been developed in o ur lab 5 . We have rai sed antiPEPC (A 111aranthus) antibodies in rabbit and used
anti-PEPC anti serum to chec k th e cross-reac tivity
with PEPC o f A /t emanthera species.
''*Correspondent author
a, p,J0 uohyd.crn et.in
Fax:
+9 1-40-JOIOI45 :
E-mail :
In contrast to the extensive literature on purifica1
tion and storage of PEPC -35 , the studies on immunolog ical properti es of PEPC are quite limited . Since
the topic is of genera l interest, a comprehensive study
has been attempted, by including a few more C 3 and
C 4 plants. Th e photosynthetic status of C r and C 4
plants and C 3-C 4 intermed iates used in thi s study has
68
already been estab li shed - .
It is well known th at
there is a sig nificant difference between PEPC of Cr
dicots and C~- monocots2.3. We have therefo re inc luded in the test plants, a few monocots as we ll.
Anti-serum prepared against PEPC of maize (a mo nocot) was also used to confirm the differe nces, if any .
between monocots and dicots. Finally, the crossreactivity was confirmed by us ing anti-PEPC antibodies rai sed against the e nzyme from Afte manthera
pungens (a C 4 -s pecies) .
Materials and Met hods
Plant lllaterial- The detail s of the plant material
and th e ir mode of multipli ca ti o n/growth have been
7
reported earlier ·'.
644
INDIAN J EXP BIOL, JULY 200 1
Experimental methods-Assay and purification of
PEPC from leaves of Amaranthus or Alternanthera
were done as per the procedure described elsewhere 5.
Anti-PEPC antiserum was rai sed in 6 month-old
white rabbits, as per the principles described by
9
Nimmo et a/. . Pre-immune serum was co ll ected from
ear-vein of the rabbit. Then, 0.5 mg of purified PEPC
in 500 f.lL, emulsified in equal volume (500 J.lL) of
50% Freund's complete adjuvant, was injected subcutanenously at about 10 sites. Four weeks later, the
animal was given (through subcutaneous injections) a
booster dose of 0.25 mg in 250 f.lL of purified enzyme, emulsified with equal volume (250 f.,lL) of 50%
Freund's incomplete adj uvant. After 2 weeks, blood
was collected from the ear vein. The blood was allowed to coagu late and the antiserum was collected
by centrifugation at I 0,000 g for 30 min. The antiserum was split into several small aliquots and stored at
-20 °C.
The anima l was again inj ected with 0.25 mg in 250
f.lL of enzyme emu lsified with equal vo lume (250 f.,lL)
of 50% Freund's incomplete adjuvant. Blood was
collected after 6 to 8 weeks. Anti-PEPC antiserum
was collected - as described above by centrifugation
and stored in multiple aliquots.
Ouchterlony double-dimensional diffusion-Specificity of PEPC antiserum was checked by using 1%
(w/v) agarose gels prepared on g lass slides. Five wells
(0.5 em diam) were punched in agarose gels, with the
help of a sharp gel-puncher. One well was in the center and was surrounded by four wells located symmetrically in the outer ring. A graph sheet was placed
below the glass slide, so as to achieve symmetry and
precise distance between well s.
In the center-well, the purified PEPC of leaf ex tract
from A. hypochondriacus (or other plants) was
loaded. Different dilutions of crude anti-PEPC antiserum ( l/10 to 11100) was loaded into other four wells
in the outer ring. The precipitin band was observed
within 24 hr of incubation at 10°-12 °C. The reaction
was stopped by washi ng the gel several times with
0.9% (w/v) NaCI , to remove unbound proteins. Preimmune serum showed no cross-reaction (i.e., no precipitation) with either purified PEPC or leaf extracts
of A. hypochondriacus . The cross-reactivi ty of PEPC
from A. hypochondriacus with other species was examined by loading purified enzyme/crude extracts of
various C3, C 3-C4 and C4 dicot and C4 monocot species
in the outer wells and center well was filled with an tiPEPC antiserum.
Western blotting--Cross-reactivity of PEPC between C 3, CrC4 and C4 plants was evalu ated through
Western blots 10 , after transferring electrophoreticall y
the proteins from the gel onto the polyvinylidene difluoride (PVDF) membranes 11 •
Proteins in the leaf extracts were separated by I 0%
SDS-PAGE. The proteins were transferred on to
PVDF membranes (immobi lon-PC from Millipore,
procured from Sigma Chemicals Co., USA). The gel,
PVDF membranes and chromatography papers were
soaked in transfer buffer (25 mM, lrris-HCII 192 mM,
g lycine; pH 8.3 and 20% (v/v) methanol) for 30 min.
The gel and membranes were sandwiched between
filter papers (three on each side) saturated wi th the
buffer and blotted using a semi-dry blotter (LKB 2117
Multiphor) for 3 hr. A constant power of 90 volts was
suppli ed (through Atto Di gi-power SJ-1081).
The transfer of proteins was confirmed by Ponceau's staining [0.2% (w/v) Ponceau's stain and 3%
(w/v) TCA]. Ponceau's stain was removed by repeated washing with distilled water. The membranes
were blocked with 5% of non-fat milk powder in Trisbuffered saline (TBS) containing 25 mM, Tri s-HCI ,
pH 7.5 and 150 rnM, NaCI. Blocking was necessary to
saturate the non-specific binding sites. The blocking
was allowd for 1 hr at room temperature with constant
shak ing.
The blocked membranes were probed for 1 hr with
antiserum of anti-PEPC (from A. hypochondriacus or
Alternanthera pungens or Zea mays) antiserum, diluted 1:500 and 1:400 respectively, in blocking so luti on. The blotted membranes were washed three times
( 15 min each wash) with TBS and incubated with
anti-l gG alkaline phosphatase conjugate (I :7500) for
1hr and washed three times. The washed blot was developed with 33 f.lL of 5-bromo-4-chloro-3indolylphosphate (BCIP; 50 mg mL- 1 stock so luti on)
and 60 f.,lL of p-nitroblue tetrazolium chloride (NBT)
(50 mg mL-1 stock so luti on in 10 mL of 16 mM TrisHC l, pH 9.5), 4 mM NaCI and 0.2 mi\t! MgC1 2.
Results and Discussion
In contrast to the extensive literature on purification and storage of P_EPC, the studi es on immunological properties of PEPC are quite limited. An attempt
has, therefore, been made to study comprehensively
the immunological properties of PEPC purified from
A. hypochondriacus, a NAD-ME type C 4-dicot plant
and assessed its cross-reactivity with a few other Cr
and C4-type dicots and C 4-monocots.
Antibodies were raised in rabbits against PEPC
purified from the leaves of Amaranthus lzypochon-
645
GAY ATHRI eta/.: IMMUNOLOGICAL CHARACTERISTIC OF PEP CARBOXYLASE
driacus. The antibody showed a titre value of 11100
against purified PEPC from leaves of A. hypochondriacus as well as PEPC in leaf extracts of A. hypochondriacus or Altemanthera pungens (data not
shown).
The specificity of PEPC-anti serum was checked
initially by immunoprecipitatio n. The leaf extracts
were incubated with anti-PEPC antiserum and incubated overnight to precipitate down the PEPC-protein.
There was a marked decrease in PEPC activ ity on
treatment with anti-PEPC antiserum, presumably due
to the precipitation of PEPC from the supernatants
(Fig. 1). In controls, when leaf extracts were treated
with non-immunised rabbit serum , there was no
change in PEPC activity in the supernatants, and obvious ly PEPC was not precipitated from the super-
natants. An examination on SDS-PAGE (10%) confirmed the presence of PEPC in the precipitate. Level
of PEPC-protein in the precipitate increased as th e
amount of PEPC antiserum increased (Fig. 2). Thi s
indicated the antiserum prepared was fairly specific to
PEPC protein even in crude leaf extracts. Sugiyama
et a/. 12 have shown the specificity of the antiserum
prepared against purified maize PEPC by immunotitration with crude extracts of maize PEPC.
Single radial immunodiffusion was performed to
quantitate the amount of PEPC protein in crude leaf
extracts usi ng ant i-PEPC antiserum of A. hypochondriacus. There was a proportionate increase in diameter of the ring with the increase in antigen concentration .
Using ELISA, it was possible to quantitate low
levels (ng) of PEPC protein in crude leaf extracts of
A. hypochondriacus, as indicated by linear relationship between immuno-precipitate-linked absorbance
and PEPC protein in leaf crude extracts. In contrast,
)..lg amounts of PEPC protein was required for detecting PEPC in pea leaf extracts indicating that the crossreactivity of C 3 dicot PEPC with C 4-PEPC was less
than that in C 4 dicot PEPC.
0.20 .--~._----t.._---tl>--------Pre-immune serum
?
·o:;
0
2
.~
biJ
C<l
E
tl
0.15
D.
u .Ec:
c...
0. 10
[.I.J
c... 0
Anti-PEPC antiserum
E
3
A
0.05
8
20 40 60 80 100 20 40 60 80 100
Anti -P EPC a nti se rum (Ill)
Fi g. 1- lmmunoprecipitation o f PEPC extracted from illuminated
leaves. To a fixed vo lume of leaf ex tract, containing 0.2 unit s of
PEPC, variab le vo lumes of anti-PEPC anti serum or preimmune
serum (0-100 Ill) were added and th e mixtures were left overnight
at 4°C. After centrifugatio n the supernatant was checked for
PEPC activity. The contro l sa mpl es were treated with no nimmuni sed se rum.
A
Fig. 2-PEPC from leaf extrac ts of Amarantlws hypoclwndriacus
after precipitation by the addition of 20-100 IlL of anti-PEPC
antiserum. The pellet was washed and exam ined by SDS-PAG E
for the presence of PEPC. PEPC bands were observed o nl y o n
precipitation with anti-PEPC antiserum (A). In controls (preimmune se rum , set 8), no PEPC band was observed indi cating
that precipitation of the enzyme did not occur.
c
B
1
2
4
3
1
2 1
2
4
3 4
3
Fig. 3---0uchterlony do uble-diffusio n, to assess the cross-reactivity of PEPC in leaf extracts of selected Cr. C 4- and C 3-C4 intermediate species (including those of 1\/temalllhera). The centre wells contained the anti-PEPC anti serum (raised against purified PEPC from 1\mara/1/hus
hypochondriacus, C4 -plant, Amaranthaceae). The o uter well No.I contained the leaf extracts of A. hypochondriacus. The outer wells 2 to 4
contained in the given order. (A)- Leaf extracts of C 4 plants- Altemanthera pungens (dicot), Zea mays (monocot) and Amaranthus viridis
(dicot): (8)- Leaf extracts of C 3 plants- Altemanthera sessilis (dicot), Commelina benRhalensis (monocot), Pisum sativum (dicot): (C) - I ,eaf
646
IND IAN J EX P BIOL, J ULY 200 1
Cross reactivity of PEPC from A. hypochondriacus,
a C4 dicot, with PEPC in leaf ex tracts of C 3 or C4
mono- and dicots was exa mined by Ouchterl ony double-diffusio n (Fig. 3). The anti-PEPC anti serum,
raised agai nst PEPC of A. hypochondriacus, showed
very strong reacti on (as indi cated by the prec ipitin
band) with Amarantlws viridis and A /t ernan/hera
pungens (C 4 -dicots). On the other hand, th e same
anti-PEPC an tiserum, showed a faint reaction with
PEPC fro m Zea mays, a C4 monocot (Fig. 3). This
sugges ted th at PEPC fro m A. hypochondriactts had
only limited identity with the tert iary structure of
PEPC from either Zea mays (C4-monocot) or C3-dicot
or C_,-C4 intermediates. Iglesias et a/. 13 have observed
through the Ouchterlony technique, that PEPC of Zea
mays
ex hibited only limited cross-reacti on and
thereby depicted partial iden tity with anti-PEPC antiserum raised against PEPC purifi ed fro m A. viridis.
Di sti nctness of C4 dicot-PEPC from th at of C4 monocot species was furth er confirmed by Western blo t
ana lysis.
Although, Ouchterl ony double diffusion did not
show
much
cross-activity
of PEPC from
A. hvpochondriacus, a C4 -dicot wi th C3 plants or Cr
c-1 intermediates, it co uld be also beca use of limited
sensiti vi ty of Ouchterlony techniq ue. Since the immune reaction can be ampli fied wit h Western blot, we
checked the cross-react ivity with c3 and c_,-c4 intermediate spec ies with th at of PEPC fro m either (Amaranthus) a C4 -dicot and mai ze (C 4-monocot) plants
using anti-PEPC anti serum. When probed with the
anti -PEPC anti serum aga inst PEPC of A. hypochondria cus, significantl y pos itive cross-reactivity was
obtained (i.e. , intensely stai ned blots) with PEPC in
leaf extracts of three C4 -d icot plants, A . hypoclwndriac us, A. viridis and A/tem anthera pungens
(Fi g. 4a). In case of these C4-dicots, th e leaf ex tracts
co ntai nin g protein (20 fl g) were loaded on the gel for
visual appearance of blot. PEPC in the leaf extracts
of three monocots (Zea mays, Sorg hum bico/or, and
Pennisetwn) could also be visualized by Western blot
using anti-PEPC antiserum agai nst C4-dicot PEPC
(Fi g. 4b). However, the protein to be loaded onto the
gel in case of C4-monocots (30 fl g of protein) was
more than that (20 fl g protei n) needed in case of C4dicots. Such need fo r hi gher levels of C4-monocot
protein than th at of C4 - clicot protei n for similar intensity on Western blots, suggested th at th e PEPC of C4dicot was immunologicall y simil ar yet distinct from
that of C4 -monocot.
Similarly, probing with PEPC-antiserum (agai nst
of PEPC of Am aranthus) could detect the PEPC in
leaf ex tracts of C 3-C4 intermediates (Parthenium hysteroplwrus, A /tern anthera ficoides, A /ternan/h era
tenet/a; Fig. 4c) as well as that of C 3 species (Pisu111
sativum, Comme/in a bengha/ensis. A /ternanth era ses'.si!is). However, the intensity of protein bands in case
of CrC 4 intermed iate species was stronger th an th at of
C3 species (Fig. 4d). PEPC-protein in leaf extrac ts of
2
1
3
110kD -+-
2
1
110k0~
I
3
~
·•·
ta
1
2
3
1
2
3
110 kD -+-
100kD -+-
11 0 kD -+-
100 kD -+-
•
Fi g. 4--Western blots of PEPC in leaf ex trac ts of selec ted C 4 - and
C 3-plants and CrC4 intermedi ates. The blots were pro bed with
anti-PEPC anti serum (raised against PEPC o f Amaranthus hvpuchondriacus, a C 4 -dicot). (A)- C 4 -dicots [Lanes I to 3 in the given
order: A. hypochondriacus, Amaranth us l'iridis, Alten w nrhera
pw1gens]; (8)- C 4 -monocot [Lanes I to 3: Zea mays. Sorghum
bico/or, Pe1111isetum glaucum]; (C)-, Cr C 4 intermedi ate dico ts
[Lanes I to 3: Parthenium hysterophom s , Altem anthera ficoides.
Altemanthera renella ]; (0)-Crplants [Lanes I to 3: Pi.\'11111 sari·
vwn, Alternanthera sessilis (both dicots ), Crmunelina benglwlensis (mo nocot)]. The location of PEPC is indicated by an arrow .
The amoun t o f pro tein loaded in each lane was 20 J..lg in case o f C4
di co ts and 30 J..lg in a ll ot her cases.
647
GA YATHRI eta/.: IMMUNOLOG ICAL C HA RACTE RI STIC OF PEP CA RBO X YLASE
C4-dicot spec ies was evident when probed with onl y
antiserum against PEPC of A. hypochondriacus, but
not of maize (Figs. 4a, Sa). Antiserum against maize
PEPC co uld react and showed up onl y the maize enzyme on the gels (Fig. Sa), but not of C4-dicots nor of
3
2
1
110klJ ---.
1
2
3
1 10 kD - .
1 00 kD ___.
:
1
2
3
C 3-C 4 intermediates (Fig. Sb,c).
Immunological distinctness of PEPC from CrC4
intermediates and C4 -dicots was further confirmed in
th e experi ments involving the use of anti-PEPC antiserum raised agai nst th e enzy me of Altemanth era
pungens (Fig. 6). When probed with thi s an ti-P EPC
(of Alternanthera) anti-serum, the strongest reacti on
was with PEPC of Amaranthus, or A. pungens (C4dicots) followed by A. tenella and A. ficoides (CrC4
intermediates), and then A. sessilis (C3 species). Least
immunoreaction was with PEPC of maize (C4monocot).
We noticed that at least two bands of PEPC appeared on Western blots with leaf extracts of C 3 and
C3-C4 intermediate spec ies indi cati ng the ex istence of
different isoforms (100 and 110 kD) of PEPC. In C4
species, onl y a single band was obtained on I0% of
SDS-PAGE gels or immunoblots. particul arl y when
loaded with 30 1-1 g or more of protein. Pattern of double-bands (with leaf extracts C 3 or C 3-C4 intermediates) after immunoblotting remained unaltered even in
presence of protease inhibitor like PMSF during extraction.
2
11 0 kD ---.
1 ~1 -
I 00 k l ) ___.
Fig. 5-Weste rn blots o f PEPC in leaf extrac ts of selec ted C 4-, or
C1-species and C 3-C 4 intermed iates. Th e blots were probed with
anti-PEPC a nti serum (raised aga inst PEPC of Zea 111ays, a C 4mon ocot). (A)-C 4 plants [Lanes I to 3 in the given order: A. hypoclwndriacus, A/terna/1/hera fJIIIIfiells , Zea 11/0}'s] ; (B) c , -C4
intermediate dicots [Lanes I to 3: Partheni11111 hysterophoms,
Alternanthera .ficoides, Alternanth era tenella] ; and (C)- C,-plan ts
I Lanes I to 3: Pisu/11 sativu111, Altern amhera sessilis (both dicots),
Colll/llelilw benghalensis (monocot) i. Fu rth e r de tai ls are as in
Fig. 4.
3
4
-- ---
5
6
7
-·
Fig. 6-Western blots of PEPC in leaf ex tracts of C 4- and . C.1 ·
plants and C 3-C4 intermediate species of Alternanthera, compared
to th e reactivity of pea, maize o r A111arantlws. Th e blo ts we re
probed w ith anti-PEPC ant iserum (ra ised against PEPC o f AlterIIOiilhera pungens, a C 4-dicot). [Lanes I to 7, in the g ive n order:
Pis11111 sativwn (C 3-dicot), Alternamhera sessi/is (C 3 dicot ). Alternan/hera ficoides (C 3-C4 intermediate dicot ), A/ternan/hera tenella (C 3-C 4 intermediate dicot), A/ternan/hera pungem (C 4 elicot), A111aranthus hypochondriacus (C 4 dicot) and Zea 11wys (C4
mo nocot) . The locati o n of PEPC is indicated by an a rrow . The
a mo unt of protein loaded in each lane was 20 Jl g. Further detail s
are as in Fig. 4.
Tab le 1-A summary o f the observati o ns o n the cross-reac ti vity of PEPC in leaf ex tracts of C 3 , C 4 and CrC 4 inte rmed iate plants
aga inst the antibod ies rai sed aga inst PEPC o f A111aramhus hypochondriacus o r maize.
Species/Photosynthetic type
Antibodies raised aga inst PEPC from
A111arantluo- hypochondriacus
A111amnthus hvpoclwndriacus (C 4 )
Zea 111ays (C4)
Sorghu111 bicolor (C 4 )
A/ternan/h era pungem (C 4 )
A. tenella (C1-C4)
A. ficoides (C, -C4)
A. sessilis (C 3 )
Pisu111 sativ11111 (C 3 )
Zea lllays
+ +++
<+
++++
++
++ ++
++
<+
++++
++
<+
++
<+
+
<+
+
<+
·+ · indi cat es a posi ti ve reac ti o n. The se nsitivity of reactio n is arbitraril y rep rese nted by the number of'+' signs
648
INDIAN J EXP BIOL, JULY 200 t
Appearance of two or th ree PEPC bands on Western blots indicating the presence of isoforms in c3
14
have been noticed in ex tracts from c3 plants
and
5
6
CAM pl ants' ·' . Wiegend and Hinch a (1992) 15 have
observed only one PEPC band with C 4 and CAM species while two bands appear o n immunoblots of extracts from c3 pl ants using anti serum against maize
PEPC. During Western blott ing, only one PEPC band
in mesophyll cell protoplasts, two PEPC bands in
mesophyll tissue preparations, and three in guard cell
protoplasts have been fo und earli er 17 . Epidermal ti ssue of Vic ia faba has shown two bands of PE PC
cross-react ing with a ntibodies against K. daigremontiana1 8. Slocombe et a/. 16 have also shown two or
th ree bands appearing in c3 performing M. crystallillU/11 using antisera direc ted against PEPC from CAMperforming K. daigremo11.tiana . Simi larly , presence
of two PEPC bands in germ in ating coty ledons of
Ricinus communis on probing with antiserum against
maize leaf PEPC have also been observed earlier 19.2°.
Based on the Ouchterlony double diffusion and
Western blot ana lys is (summarized in Table I), possibly PEPC from C4-dicots was distinct from C 4 monocots (or CrC 4 intermediates or C 3 plants), as
indicated by cross reactivity. Similar suggestion has
been made earlier based on comparison of e DN A se2
quences of PEPC . Simil arly PEPC of C 3-C 4 intermediates ex hibited a dist inct in termediacy in term s of
immunological cross-reactivity between C 4 and C 3species. This is the first report on such immunological distinctness of PEPC from CrC 4 intermediates
within the genus of A ltemanthera . It has been suggested that Alternantlzera can be another good model
to study the molecular nature of PEPC, like Flaveria.
Acknowledgement
This work was supported by a grant from Yolkswagen-Stiftung (No. 1171-371) for a collaborative
project between Prof. Peter Westhoff (U niversiUit
DUsseldorf, Germany) and Prof. A.S . Raghavendra
(University of Hyderabad, India).
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