Plant CellPhysiol. 38(8): 928-934 (1997)
JSPP © 1997
Characteristics and Physiological Function of NADP-Malic Enzyme from
Wheat
Paula Casati 1 , Claudia P. Spampinato' and Carlos S. Andreo 2
Centro de Estudios Fotosinteticos y Bioqui'micos, UniversidadNational deRosario, CONICET, Suipacha531, 2000Rosario, Argentina
Kinetic and structural properties of NADP-malic enzyme (NADP-ME, EC 1.1.1.40) purified from stems and
roots of wheat (Triticum aestivum), along with the possible
physiological role of the enzyme were examined. Enzyme
purification from stems sequentially involved precipitation
with crystalline ammonium sulfate, anion-exchange, affinity and size exclusion chromatographies, while anion-exchange chromatography was omitted for the enzyme purification from roots. SDS-PAGE of the purified enzyme
showed a single protein band with a molecular mass of 72kDa. Enzyme activity was dependent on the presence of a
bivalent metal cation, Mg2+ or Mn 2+ . Binding characteristics of each metal ion suggest the existence of at least two
different binding sites with distinct affinities. Nonetheless,
activity response to NADP + and L-malate exhibited
Michaelis-Menten behavior with Km values of 37 and 960
ftM, respectively. The amount and activity of NADP-ME
were increased by GSH, cellulase and macerozyme. From
these results we suggest that NADP-ME of wheat could be
implicated in defense-related deposition of lignin.
Key words: C3 plant — Cellulase — GSH — Kinetic Properties — Macerozyme — Triticum aestivum.
a cytosolic isoform also functions as a decarboxylase of
malate to donate CO2 for the Calvin cycle (Ting 1985).
Along with this function NADP-ME is thought to act in
conjunction with phosphoenolpyruvate carboxylase to regulate intracellular pH (Davies 1986). In this way, regulated
changes in the activity of this isoform permit the diurnal
fluctuation of malic acid (Spalding et al. 1979, Ting 1985).
Apart from these specialized uses, NADP-ME seems to
fulfill diverse housekeeping functions because of its universal presence in many different tissues. These other, nonphotosynthetic roles are less well understood. They may be
anaplerotic, providing NADPH and pyruvate in biosynthesis, or catabolic, participating in respiration of these products (Edwards and Andreo 1992).
Currently, most information about NADP-ME is
related to C4 plants (Edwards and Andreo 1992). In this
case, physical and kinetic properties, occurrence, function
and regulation are known. However, reports on the enzyme from C3 plants are scarce. Therefore, the aim of the
present paper was to purify and characterize the physical,
immunological and kinetic properties of NADP-ME from
wheat, along with its possible physiological role.
Materials and Methods
Chemicals—NADP+, L-malic acid, Tris, BSA, molecular
weight standards, Freund's adjuvant, alkaline phosphatasetagged goat anti-(rabbit IgG) IgG were from Sigma Chemicals
Co., U.S.A. Sephacryl S300 HR was purchased from Pharmacia,
Sweden. Ampholine carrier ampholytes were from LKB-Pharmacia. All other reagents were of analytical quality.
Plant material and enzyme purification—Wheat (Triticum
aestivum) plants were grown outdoors (approximately 14 h photoperiod and 25°C day/15°C night temperature regime). Stems
from 15-week-old plants (about 250 g) were cut and used within
the day. The material was washed and chopped into pieces, then
suspended in 300 ml of a extraction buffer (buffer A) containing
100 mM Tris-HCl (pH 7.3), 10 mM MgCl2, 2 mM Na 2 HPO 4 , 1
mM EDTA, 20% (v/v) glycerol and 10 mM 2-mercaptoethanol.
The stems were homogenized using a blender. The homogenate
was filtered through cheesecloth and centrifuged at 9,000 x g for
15 min. To the supernatant, crystalline ammonium sulfate was
gradually added up to 30% saturation. After centrifugation at
9,000 xg for 30 min, the supernatant was brought to 60% saturation and centrifuged.
The resulting precipitate was dissolved in 20 ml of purification buffer (buffer B) containing 50 mM Tris-HCl (pH 7.3), 5 mM
MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol and 10 mM 2-mercaptoethanol and passed through a column of Sephadex G-75
(2.5x40 cm), previously equilibrated with buffer B. The eluate
The NADP-malic enzyme (NADP-ME; L-malate:
NADP oxidoreductase [oxaloacetate decarboxylating], EC
1.1.1.40) catalyses the oxidative decarboxylation of Lmalate to yield pyruvate, CO 2) and NADPH and requires a
bivalent metal ion as an essential cofactor (Edwards and
Andreo 1992). The enzyme acts in many different metabolic
pathways in plants. In C4 species, an abundant isoform of
NADP-ME is localized in the chloroplasts of bundle sheath
cells (Maurino et al. 1997), where it releases CO2 to be used
in carbon fixation by ribulose-l,5-bisphosphate carboxylase (Edwards and Huber 1981). This pathway eliminates
the photorespiratory loss of CO2 that occurs in most plants
in which O 2 can enter the phosphoglycolate pathway via
ribulose-l,5-bisphosphate carboxylase. In CAM plants,
Abbreviations: BSA, bovine seric albumin; CAM, Crassulacean acid metabolism; FPLC, fast protein liquid chromatography; NADP-ME, NADP-malic enzyme.
1
Both authors contributed equally to this work.
2
Corresponding author: fax 54-41-370044, e-mail csandreo®
agatha.unr.edu.ar
928
NADP-malic enzyme from wheat
was then applied to a column of DEAE cellulose (2 x 20 cm) previously equilibrated with the above buffer. After washing with 3 volumes of buffer B at 30 ml h ~ \ the enzyme was eluted with 200-ml
linear gradient of NaCl (0-300 mM) at 60 ml h~'. The fractions
containing NADP-ME activity were pooled and the protein was
precipitated with solid ammonium sulfate at 60% saturation.
The precipitate was collected by centrifugation, dissolved in
5 ml of buffer containing 50 mM Tris-HCl (pH 7.3), 5 mM MgCl2,
0.1 mM EDTA, 5% (v/v) glycerol and 10 mM 2-mercaptoethanol
(buffer C) and passed through a column of Sephadex G-75 (2 x 20
cm). The fractions containing NADP-ME activity were pooled
and applied to an Alfi-Gel blue column (1.5 x 10 cm) connected to
a Pharmacia FPLC system pre-equilibrated with buffer C. The column was washed with 3 volumes of buffer C and the NADP-ME
was eluted with a linear gradient of NaCl (0-300 mM). The fractions containing the enzyme activity were eluted with 220-240 mM
NaCl. Finally, the enzyme was applied to a Superose 12 column attached to the FPLC system and pre-equilibrated with buffer C.
Purified NADP-ME was stored at - 20°C until use, typically within a week.
Wheat roots were obtained from plants grown 15 days in vermiculite. The purification procedure used was similar to the one
described above with the following modifications: (i) buffer A contained 1 mM phenyl-methylsulfonyl fluoride in addition; (ii) crude
extract was precipitated with crystalline ammonium sulfate at 3070% saturation; (iii) the Sephadex column was equilibrated and
eluted with buffer C; (iv) the chromatography on DEAE-cellulose
column was omitted.
Protein measurement—Total protein was determined by the
method of Lowry et al. (1951) or alternatively by the dye-binding
method of Sedmak and Grossberg (1977). BSA was used as standard.
Enzyme assay—NADP-ME reaction was determined spectrophotometrically at 30°C by monitoring NADPH production at
340 nm. The standard assay medium contained (unless otherwise
stated) 50 mM Tris-HCl (pH 7.5), 0.5 mM NADP + , 10 mM Lmalate, 10 mM MgCl2 and 20-30 fig of malic enzyme in a final volume of 1 ml. One unit of enzyme activity is defined as the amount
of enzyme resulting in the production of 1 fimol of NADPH per
minute.
Since metal-ligand chelating complexes are not reactants for
the NADP-ME reaction (Iglesias and Andreo 1990), the concentration of each substrate or cofactor was corrected for the concentration of the chelate complexes (Grover et al. 1981). The following
dissociation constants were used in the correction: Mg-malate,
28.2 mM; Mg-NADP, 19.1 mM; Mn-malate, 5.75 mM; MnNADP, 1.1 mM (Grover et al. 1981).
The experimental data were fitted to the Michaelis-Menten
equation by a non-linear least-squares regression computer kinetics
program (Brooks 1992).
Determination of native molecular mass—The molecular
weight of native NADP-ME was determined using a column of
Sephacryl S300 HR connected to a Pharmacia FPLC system.
Equilibration and elution were performed at 25 °C with 50 mM
Tris-HCl (pH 7.0 or 8.0) at a flow rate of 1 ml rnin"'. The column
was calibrated with thyroglobulin (M, 669,000), apoferritin (M,
443,000), yS-amylase (A/r 200,000), BSA (A/r 66,000), and carbonic
anhydrase (Mr 29, 000). The void volume was determined with
blue dextran.
Linear correlation was obtained when logarithm of M, was
plotted against Kav. This parameter is defined as: K av =(V e —V o )/
(Vt—Vo) being: Vc the elution volume, Vo the void volume and V,
the total volume of the packed bed.
929
Electrophoresis and isoelectricfocusing—Native and denaturing PAGE were performed according to Laemmli (1970) using the
Bio Rad mini-gel apparatus.
The native PAGE (6% w/v final acrylamide monomer concentration) was run at pH 8.0.
The final acrylamide monomer concentration in the denaturing gel was 10% (w/v) for the separating gel and 5% (w/v) for the
stacking gel. The following standard proteins were used for the
determination of the subunit molecular mass: BSA (M, 66,000),
ovalbumin (Afr 45,000), glyceraldehyde 3-phosphate dehydrogenase (Mr 36,000), carbonic anhydrase (Mr 29,000) and trypsin inhibitor (Mr 20,100). Coomasie Brilliant blue G-250 was used as a
staining reagent for the detection of protein bands.
A 5% (w/v) polyacrylamide gel isoelectric focusing (pH 3-9)
was performed using an LKB 2117 Multiphor system. Samples
were applied to the gel surface on sample application pieces
(LKB). The gels were run for 3 h at 6°C (constant power: 0.6 kV).
NADP-ME was detected in native and isoelectrofocusing gels
by incubating them in the standard reaction mixture for assaying
malic enzyme containing 0.01% (w/v) nitrobluetetrazolium chloride and 0.05% (w/v) phenazinemethosulfate (Gabriel 1971).
Antibody production and
immunoblotting—Antibodies
against NADP-ME were obtained by immunization of two New
Zealand white rabbits with 250/ig of the purified protein emulsified in Freund's complete adjuvant, in subcutaneous (200fig)
and intramuscular (50^g) injections. Booster injections (100
fug) of the same protein mixed with incomplete adjuvant were administered subcutaneously after 2 and 4 weeks. Fifteen days after
the final injection the rabbits were anesthetized and the blood was
collected by cardiac puncture. The crude antiserum was frozen in
liquid N2 and stored at - 80°C with 0.04% (w/v) NaN3. For immunoblotting, the serum was affinity purified against NADP-ME as
described by Plaxton (1989). Non-immunized serum showed no
cross-reaction with NADP-ME.
Immunoblotting was performed according to Burnette
(1981). Purified wheat malic enzyme (3 fig) or crude extracts from
wheat leaves (30 fig) were run on a denaturing PAGE, transferred
to nitrocellulose membranes and probed with purified anti-(wheat
stem NADP-ME). Antigenic polypeptides were visualized using
an alkaline phosphatase-tagged secondary antibody.
For quantitation of the enzyme, densitometric analyses were
performed with Quantiscan program (Microbial System Ltd). In
integrating the peak areas, each peak was determined in triplicate
to minimize systematic errors.
Stress treatments—Wheat seeds were germinated on moist vermiculite. After 15 days, the fully developed leaves were detached
and placed vertically in a flask containing destilled water in the
absence (control) or presence of various compounds. Incubation
of detached leaves was conducted at 20°C under a 14 h photoperiod. At the indicated times the leaves (200 mg) were immediately
frozen in liquid N2 and stored at — 80°C prior to enzyme extraction. Extraction was achieved by grinding leaves with a mortar in
liquid N2 and resuspending the powder in 0.5 ml buffer A. The extract was centrifuged (15,800 xg for lOmin) and the supernatant
obtained was used for malic enzyme assay.
In all the cases, the data were statistically analyzed with
INSTAT program from Sigma Chemicals Co., U.S.A.
Results and Discussion
Isoelectric focusing—Crude enzyme extracts obtained
from maize and wheat leaves were electrophoresed and
then assayed for malic enzyme as described in Materials
930
NADP-malic enzyme from wheat
A
B
C
0
MW A
B
C
D
5.7
66
6.6
45
29
Fig. 1 Polyacrylamide gel isoelectric focusing revealed for malic
enzyme activity. Crude extracts (30 fig of total protein) of maize
leaves (A), wheat leaves (B), wheat stems (C) and wheat roots (D)
were applied to each lane.
and Methods. The results indicate that maize NADP-ME
shows a pi of 6.6±0.2 while the value for the wheat enzyme is 5.7±0.1 (Fig. 1A, B). The same technique applied
to the extracts from different tissues of wheat (leaves, stems
and roots) revealed the presence of NADP-ME (Fig. IB, C,
D) with the same value of pi. This result suggests the presence of only one kind of NADP-ME in C3 tissues.
Enzyme purification, molecular weight and immunological properties—NADP-ME from wheat stems and
roots was purified by conventional techniques about 21-
24
mk
•
. •
.
>~-
•••
Fig. 2 Denaturing PAGE of the purified malic enzyme from
wheat stem (3 fig) (A, D), wheat root (3 fig) (B) and maize leaf (6
fig) (C) stained for protein (A, B, C) and revealed by immunoblot
analysis probed with anti-(wheat stem NADP-ME) (D). The numbers on the left side of the figure are molecular weight of the
markers, given in kDa.
and 10-fold, respectively, to a final specific activity of 0.98
U m g " ' . As in the case with the non-photosynthetic enzyme from maize (Table 1), the purified wheat NADP-ME
subjected to denaturing PAGE shows one polypeptide of
72 kDa (Fig. 2A, B) which reacted with the specific antibodies (Fig. 2D). However, the size of the monomer of the
Table 1 Characteristics of photosynthetic and non-photosynthetic NADP-ME of maize and wheat
Maize NADP-ME
Photosynthetic tissue
Structural and
kinetic property
Molecular Weight
Isoelectric Point
Optimal pH
Optimal T (°C)
Activation energy (kcal mol" 1 )
61.3"-*
6.6
7.8-8.4*
53*
9.5*
pH 8.0
VmM (U ing" 1 )
+
Km NADP (jiM)
Km Mg (mM)
Km Mn G*M)
Km Malate (mM)
K, Malate (mM)
30.9 c
8.6C
0.23 d
1,100''
0.18'
ni'-'
0.05 d
' Rothermel and Nelson (1989).
* Asami et al. (1979).
c
Spampinato et al. (1991).
" Drincovich et al. (1991).
' Spampinato et al. (1994).
1
nd: not determined; ni: no inhibition was observed.
* Maurino et al. (1996).
* Scagliarini et al. (1988).
pH 7.0
8.1 d
nd'
0.94 d
1,830"
0.088'
4.3'
0.11"
1.2"
Non-Photosynthetic
Wheat NADP-ME
72.0
72.0*
5.4*
7.0*
nd'
nd'
55
10.0
pH7.0
pH7.5
1.10*
6.9*
0.089*
nd'
0.17*
nd'
5.7
7.2
0.98
37
0.022*
0.20
0.56
0.96
ni'
0.006
0.066
NADP-malic enzyme from wheat
photosynthetic C4 isoenzyme was estimated to be 61-63
kDa (Fig.2C, Table 1) (Rothermel and Nelson 1989,
Borsch and Westhoff 1990, Maurino et al. 1996).
The native molecular mass of the enzyme estimated by
size exclusion chromatography on FPLC (calibrated with
standard markers, Fig. 3A) at different pH values (7.0 or
8.0) was 142.5 + 3.5 kDa (Fig. 3B). It is worth to be men1000
«-- 100 -
I
4 6
8
14
10
16
Volume (ml)
\
931
tioned that this value fits to the maximum of a broad peak
(Fig. 3B) that might show the coexistence of the three different aggregation states of the enzyme (tetramer, dimer, monomer). Another result supporting this idea comes from
native gels electrophoresis (Fig. 3C) and crosslinking experiments of the enzyme subunits performed at both pH values
(Spampinato et al., in preparation).
Kinetic properties of the purified stem malic enzyme—
Preincubation of malic enzyme in the presence or absence
of the cofactor or substrates prior to assay caused no
changes in the observed kinetic properties (data not
shown). These results indicate that the enzyme purified
from C3 plants does not exhibit hysteretic properties, contrary to the results described for the C4 enzyme (Iglesias
and Andreo 1992).
Effect ofpH and temperature—The enzyme assayed in
the presence of 10 mM L-malate, 0.5 mM NADP + and 10
mM Mg2^., exhibited a broad pH/activity profile with a
maximum occurring between pH 7.0 and 7.5 (Fig. 4A). In
agreement with previous observations, the pH response of
the enzyme from wheat is like that of the one from C3 and
CAM plants rather than that from malate-producing C4
plants (Edwards and Andreo 1992).
When the enzyme activity was measured using the
standard assay system at temperatures ranging from 5 to
70°C, the maximal activity was observed at 55°C (Fig. 4B).
From a linear Arrhenius plot of the data, in a temperature
B
•T
•D
-M
Fig. 3 A, Calibration plots of the FPLC size exclusion column.
Individual proteins of known molecular mass were run in TrisHC1 (50 mM each) buffer, adjusted at pH 7.0 or 8.0. Each point is
the average of two individuals runs. Changes in pH did not alter
the apparent mass of the protein standards. From the elution volume of the proteins, Km was calculated as described under
Materials and Methods. B, FPLC elution profile of NADP-ME
after gel filtration. After 30 min of equilibration at each pH, the
purified enzyme (50 ftg in 50 /jl) was injected and run by using
Tris-HCl (50 mM each) buffer, adjusted at pH 7.0 or 8.0. C,
Native PAGE of the purified malic enzyme from maize (A) and
wheat stem (B) (3 /jg) revealed by malic enzyme activity. T, D and
M correspond to the tetramer, dimer and monomer, respectively.
M runs with the dye front.
10
20
30
40
50
60
80
Temperature (°C)
Fig. 4 A, pH dependence on the malic enzyme activity. The
assay was performed in duplicate. B, Effect of temperature on the
activity of wheat malic enzyme. The inset shows the Arrhenius
plot of the data in a temperature range from 5 to 40°C. The data
were the average of triplicate measurements.
932
NADP-malic enzyme from wheat
0.00
0.05
0.10
0.15
0.20
0.25
0.2
o.o
0.4
0.6
NADP* (mM)
Fig. 6 Effect of NADP + concentration on malic enzyme activity. The kinetics assays were carried out at saturating L-malate (10
mM) and Mg2* (10 mM) levels. The inset figure shows the Hill
plot of the data. The results are presented in terms of the calculated free NADP + concentrations (see Materials and Methods). The
assay was performed in duplicate.
or Mn 2+ concentrations at saturating concentrations of
NADP + (0.5 mM) and L-malate (10 mM) showed non[Mr,2*]"' (MM)"1
hyperbolic response with biphasic Lineweaver-Burk plots
(Fig. 5). From these results, two V^ and Km values could
Fig. 5 Lineweaver-Burk plots of malic enzyme activity as a funcbe calculated, with Mn 2+ having lower Km values and
tion of essential bivalent metal ion concentration. The kinetic
assays were performed at saturating NADP + (0.5 mM) and L- higher Vm!a/Km ratios (Table 2). These results indicate that
malate (10 mM) concentrations as described in Materials and
NADP-ME from wheat has a high preference for Mn 2+
Methods. A, Effect of Mg 2 *. B, Effect of Mn 2 + .
over Mg 2+ . Nonetheless, the physiological role for these
metal ions remains uncertain in the absence of precise data
on their relative concentration in the cell. The saturation
range from 5 to 40° C, the activation energy of the reaction kinetics were non-hyperbolic, suggesting the existence of at
was calculated to be 10,023±404 cal m o P 1 (Fig. 4B inset). least two different binding sites on the enzyme, as was previThis result is similar to the one measured with the en- ously reported for the C4 isoenzyme (Iglesias and Andreo
zyme purified from maize photosynthetic tissues (Table 1)
1990, Drincovich et al. 1991).
(Asami et al. 1979).
Substrate kinetics—The saturation curves obtained
Cation requirements—As is the case with other when the velocity of the reaction was measured as a funcNADP-ME (Drincovich et al. 1991, Edwards and Andreo tion of free NADP + (not metal-chelating concentration) in
1992), the C 3 enzyme required a bivalent metal ion for its the presence of saturating concentration of L-malate (10
activity. The saturation curves of the enzyme for Mg 2+ mM) and Mg2* (10 mM) were typically hyperbolic (Fig. 6),
Table 2 Kinetic parameters of NADP-ME using Mg2* and Mn2* as cofactors
Cation
Vm (U mg~ •)
Km (mM)
Mg2*
0.161 ±0.016
5.8xl0- 3 ±1.5xl0~ 3
27.8
0.979±0.022
2.0xl0-'±2.0xl0~ 2
4.9
Mn
2
0.407±0.011
2
5
6
6,167
4
5
1,025
6.6xl0- ±8.0xl0~
0.574 ±0.006
5.6xl0~ ±5.4xl0"
2
'max'-^m
The range of concentrations for Mg * and Mn * were 4 x 10 3-10 mM and 10 3 -l mM, respectively.
The kinetic assays were performed at saturating NADP* (0.5 mM) and L-malate (10 mM) concentrations. Each experiment was performed in duplicate.
NADP-malic enzyme from wheat
which was in agreement with a Hill coefficient of 1.0 obtained from the Hill plots (Fig. 6 inset). In this way, a low Km
value (37 ± 3 fiM) for free NADP + was calculated as it had
been observed in most studies (Edwards and Andreo 1992).
In general, there is no evidence that different isoenzymes
of NADP-ME in plants have differences in kinetics with
respect to NADP + . The high affinity and binding site
for NADP + may have been conserved during evolution
(Rothermel and Nelson 1989). It is interesting to note that
there was no activity with NAD + as it was previously
reported for the maize enzyme (Spampinato et al. 1991).
In contrast, the kinetics with respect to malate vary
from hyperbolic, to negative cooperativity, to sigmoidal
depending on the source of the enzyme and the pH of the
assay (Edwards and Andreo 1992). Our results indicated a
hyperbolic response with a Km value of 0.96 mM (Table 1).
This result is in agreement with several previously reported
values, indicating that the Km value for malate is higher in
NADP-ME from C3 and CAM species than that in the enzyme from C4 type plants (Edwards and Andreo 1992).
Physiological role—Although NADP-ME plays crucial
roles in certain types of specialized plant metabolism such
as C4 and CAM, the apparently universal presence of the
malic enzyme in plants suggests that it has a function
broader than these specialized purposes. Therefore, a variety of compounds was tested as possible inducers of NADPME.
NOf (0.8 and 40 mM), provided in a watering solution for whole plants or taken up by detached leaves, and
ABA (100 /xM), H2O2 (300 mM) and ascorbic acid (10
mM), absorbed by detached leaves, were found to be ineffective in stimulating the malic enzyme activity.
Inducibility by light was also analyzed. Plants were
grown 10 days in darkness and then exposed to white light
for periods ranging from 2 to 120 h. No difference in
NADP-ME due to an effect of the light treatment was seen.
On the other hand, cellulase (0.5mgml~') and
macerozyme (0.5 mg ml" 1 ), enzymes that degrade the cell
wall, caused a 2.2- and 1.2-fold increase in the level of
NADP-ME activity, respectively (Table 3). The induction
933
A
B
Control
GSH
Cellulase
Fig. 7 A, Denaturing PAGE of the crude extracts (30 ng of total
protein) of wheat leaves incubated 48 h in the absence (control) or
presence of GSH (10 mM), cellulase (0.5 mg m T ' ) or macerozyme
(0.5 mg ml"1) revealed by immunoblot analysis. B, Relative areas
of the integrated peaks determined by densitometric analysis of A.
Each peak was integrated in triplicate to minimize systematic errors. The figure shows a typical result.
of activity was associated with amounts of the enzyme
(Fig. 7A), indicating that the response due to the inducers
could involve de novo synthesis of the protein. The densitometric analysis showed that the levels of the enzyme
treated with cellulase and macerozyme increased 2.5- and
1.9-fold, respectively (Fig. 7B). Cellulase was reported to
be the most potent elicitor to induce lignification in wounded wheat leaves (Barber and Ride 1988). Macerozyme also
possessed elicitor activity though much weaker than cellulase (Barber and Ride 1988).
Furthermore, GSH (10 mM) was found to be another
inducer, giving a 1.4- and 1.5-fold increase in the activity
and amount of NADP-ME, respectively (Table 3, Fig. 7).
GSH, the most abundant non-protein thiol molecule in
plant cells, appears to play a key role in protection against
oxygen radicals (Alscher 1989). The level of GSH in foliar
Table 3 Effect of various compounds on the activity (U mg ') of NADP-ME from wheat
leaves
Time(h) -
Macerozyme
Treatment
Control
GSH
Cellulase
Macerozyme
0
0.048±0.003
0.048 ±0.003
0.048 ±0.003
0.048 ±0.003
24
0.060±0.019
0.056±0.012
0.086±0.016
0.059±0.016
48
0.066 ±0.002
0.093 ±0.017
0.140±0.014
0.079±0.001
Detached leaves were incubated in the absence (control) or presence of GSH (10 mM), cellulase (0.5
mg ml" 1 ) or macerozyme (0.5 mg ml" 1 ) for the indicated periods and processed as described under
Materials and Methods section. Each experiment was performed in quadruplicate.
934
NADP-malic enzyme from wheat
tissues has been shown to increase under various oxidative
stress conditions (Alscher 1989) and induce a massive and
selective transcription of defense genes encoding enzymes
of phytoalexin and lignin biosynthesis as well as stimulation of genes encoding cell wall hydroxyproline-rich glycoproteins (Wingate et al. 1988).
It has also been reported that wounding caused a
marked induction of a bean malic enzyme promoter activity in transgenic tobacco, which was further strongly enhanced upon application of stimuli related to pathogen
defense (Schaaf et al. 1995). In addition, the gene for the
bean malic enzyme exhibits novel regulatory characteristics
in that it is responsive to an elicitor for a fungal pathogen
(Walter et al. 1994), suggesting the involvement of NADPME in pathogen-related and stress-related defenses.
In conclusion, since cellulase and macerozyme caused
induction of lignification of wounded wheat leaves (Barber
and Ride 1988) and GSH stimulated several enzymes of
lignin biosynthesis (Wingate et al. 1988), we suggest that
malic enzyme of wheat (a C3 plant) could be implicated in
defense-related deposition of lignin by providing NADPH
for the two NADPH-dependent reductive steps in monolignol biosynthesis (Whetten and SederofF 1995). The
provision of NADPH for lignin biosynthesis has been attributed to the oxidative pentose phosphate cycle (Pryke
and ap Rees 1977). However, in rat liver undergoing
lipogenesis, activation of the pentose phosphate cycle is
supplemented by recruitment of NADP-ME to meet the
large need for NADPH in these particular development circumstances (Fabregat et al. 1987). A similar situation
might exist in lignin-synthesizing plants.
This work was supported by grants from CONICET and Fundaci6n Antorchas. PC and CPS are supported by CONICET
Predoctoral and Postdoctoral Fellowships. CSA is an established
Investigator of CONICET.
References
Alscher, R.G. (1989) Biosynthesis and antioxidant function of glutathione
in plants. Physiol. Plant. 11: 457-464.
Asami, S., Inoue, K., Matsumoto, K., Murachi, A. and Akazawa, T.
(1979) NADP-malic enzyme from maize leaf: purification and properties. Arch. Biochem. Biophys. 194: 503-511.
Barber, M.S. and Ride, J.P. (1988) A quantitative assay for induced
lignification in wounded wheat leaves and its use to survey potential elicitors for the response. Physiol. Mol. Plant Pathol. 32: 185-197.
Borsch, D. and Westhoff, P. (1990) Primary structure of NADP dependent
malic enzyme in dicotyledonous C* plant Flaveria trinervia. FEBS Lett.
273: 111-115.
Brooks, S.P.J. (1992) A simple computer program with statistical tests for
the analysis of enzyme kinetics. Biotechniques 13: 906-911.
Burnette, W.N. (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195-203.
Davies, D.D. (1986) The fine control of cytosolic pH. Physiol. Plant. 67:
702-706.
Drincovich, M.F., Iglesias, A.A. and Andreo, C.S. (1991) Interaction of
divalent metal ions with the NADP-malic enzyme from maize leaves.
Physiol. Plant. 81: 462-466.
Edwards, G.E. and Andreo, C.S. (1992) NADP-malic enzyme from plants.
Photosynth. Res. 31: 1845-1857.
Edwards, G.E. and Huber, S.C. (1981) The C4 pathway. In The Biochemistry of Plants. A Comprehensive Treatise, Vol. 8. Edited by Hatch, M.D.
and Boardman, N.K. pp. 237-281. Academic Press, New York.
Fabregat, I., Revilla, E. and Machado, A. (1987) The NADPH consumption regulates the NADPH producing pathways (pentose phosphate cycle and malic enzyme) in rat adipocytes. Mol. Cell Biochem. 174: 77-81.
Gabriel, 0. (1971) Locating enzymes in gels. Methods Enzymol. 21: 578604.
Grover, S.D., Canellas, P.F. and Wedding, R.T. (1981) Purification of
NAD malic enzyme from potato and investigation of some physiological
and kinetic properties. Arch. Biochem. Biophys. 209: 396-407.
Iglesias, A.A. and Andreo, C.S. (1990) Kinetic and structural properties of
NADP-malic enzyme from sugar cane leaves. Plant Physiol. 92: 66-72.
Iglesias, A.A. and Andreo, C.S. (1992) Hysteretic properties of NADPmalic enzyme from sugar cane leaves. Photosynth. Res. 31: 89-97.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227: 680-685.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. /. Biol. Chem. 193:
265-275.
Maurino, V.G., Drincovich, M.F. and Andreo, C.S. (1996) NADP-malic
enzyme isoforms in maize leaves. Biochem. Mol. Int. 38: 239-250.
Maurino, V.G., Drincovich, M.F., Casati, P., Andreo, C.S., Ku, M.S.B.,
Gupta, S.K., Edwards, G.E. and Franceschi, V.R. (1997) NADP-malic
enzyme: immunolocalization in different tissues of the C4 plant maize
and the C3 plant wheat. /. Exp. Bot. 48: 799-811.
Plaxton, W.C. (1989) Molecular and immunological characterization of
plastid and cytosolic pyruvate kinase isoenzymes from castor-oil-plant
endosperm and leaf. Eur. J. Biochem. 181: 444-451.
Pryke, J.A. and ap Rees, T. (1977) The pentose phosphate pathway as a
source of NADPH for lignin synthesis. Phytochemistry 16: 557-560.
Rothermel, B.A. and Nelson, T. (1989) Primary structure of the maize
NADP-dependent malic enzyme. /. Biol. Chem. 264: 19857-19592.
Scagliarini, S., Pupillo, P. and Valenti, V. (1988) Isoforms of NADP-dependent malic enzyme of the greening maize leaf. / . Exp. Bot. 39: 11091119.
Schaaf, J., Walter, M.H. and Hess, D. (1995) Primary metabolism in plant
defense. Regulation of a bean malic enzyme gene promoter in transgenic
tobacco by developmental and environmental cues. Plant Physiol. 108:
949-960.
Sedmak, J.J. and Grossberg, S.E. (1977) A rapid, sensitive and versatile
assay for protein using Coomasie Brilliant blue G250. Anal. Biochem.
79: 544-559.
Spalding, M.H., Schmitt, M.R., Ku, S.B. and Edwards, G.E. (1979) Intracellular localization of some key enzymes of Crassulacean acid metabolism in Sedum praealtum. Plant Physiol. 63: 738-743.
Spampinato, C.P., Colombo, S.L. and Andreo, C.S. (1994) Interaction of
analogues of substrate with NADP-malic enzyme from maize leaves.
Photosynth. Res. 39: 67-73.
Spampinato, C.P., Paneth, P., O'Leary, M. and Andreo, C.S. (1991) Analogues of NADP as inhibitors and coenzymes for NADP-malic enzyme
from maize leaves. Photosynth. Res. 28: 69-71.
Ting, I.P. (1985) Crassulacean acid metabolism. Annu. Rev. Plant
Physiol. 36: 448-453.
Walter, M.H., Grima-Pettenati, J. and Feuillet, C. (1994) Characterization of a bean (Phaseolus vulgaris L.) malic enzyme gene. Eur. J. Biochem. 224: 999-1009.
Whetten, R. and Sederoff, R. (1995) Lignin biosynthesis. Plant Cell 7:
1001-1013.
Wingate, V.P.M., Lawton, M.A. and Lamb, C.J. (1988) Glutathione
causes a massive and selective induction of plant defense genes. Plant
Physiol. 87: 206-210.
(Received January 28, 1997; Accepted May 30, 1997)