Plant Physiol.

(1978) 62, 618-621

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves

CELLULAR DISTRIBUTION OF ENZYMES AND ENERGY SOURCES FOR NITRATE REDUCTION'
Received for publication February 27, 1978 and in revised form May 10, 1978

CARLOS A. NEYRA2 AND RICHARD H. HAGEMAN

Department ofAgronomy, University of Illinois, Urbana, Illinois 61801

ABSTRACT reduction. In corn, malate is a primary product of CO2 fixation in

Time locaization of enzymes responsible for nitrate assimilation and the mesophyll chloroplasts, while hexose and starch production is
generation of NADH for nitrate reduction were studied in corn ( Zes mays
confined primarily to the bundle sheath cells (7, 12).
L.) leaf blades. The techniques used effectively separated mesophyll and
The objectives of this work, with corn leaves, were to study: (a)
bundle sheath cells as judged by microscopic observations, enzymic assays,
the relative intercellular distribution of nitrate-assimilating and
NADH-generating enzymes; and (b) the relationship between the
chloropbhyll a/b ratios and pbotochemical activities. Nitrate reductase, photosynthetic C-4 pathway and nitrate assimilation.
nitrite reductase, and the nitrate content of leaf blades were localized
primarily In the mesophyll cells, altbough some nitrite reductase was found
in the bundle sheath cells. Glutanmne synthetase, NAD-malate dehydro- MATERIALS AND METHODS
genase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glu- Plant Material. Corn (Zea mays L.) seedlings (hybrid Oh43 x
tamate dehydrogenase were found in both types of cells, however, more B 14 except where noted) were grown in Vermiculite and irrigated
NADP-glutamate dehydrogenase was found in the bundle sheath cells than daily with full strength Hoagland solution. The seedlings were
in the mesophyll cells. These data indicate that the mesophyll cells are the grown in a 24-hr regime of 14-hr light (2,500 ft-c) and 10-hr
major site for nitrate assimilation in the leaf blade because they contained darkness to 30 and 21 C, respectively. The second and third leaves
an ample supply of nitrate and the enzymes considered essential for the were excised from 10- to 12-day-old plants after 4 to 5 hr of
assimilation of nitrate into amino acids. Because the specific activity of illumination.
nitrate reductase was severafold lower than the other enzymes involved in Isolation of Mesophyll Fraction and Bundle Sheath Cells. Der-
nitrate assimilation, nitrate reduction is indicated as the rate-limiting step ibbed minced corn leaf blades were ground for 10 sec (half-line
in situ. A sequence of reactions is proposed for nitrate assimilation in the voltage) in a water jacket Waring Blendor at 0 C in grinding
mesophyll cells of com leaves as related to the C4 pathway of photosyn- medium A that contained: 0.33 M sorbitol, 50 mm Tricine (pH 8.0),
thesis. 5 mM MgCl2, I mm MnCl2, 0.25 mm K2HPO4, 2 mm Na2EDTA,
5 mm isoascorbate, 2 mm thioglycolate, and 2% (w/v) PVP-40
(Sigma Chemical Co.). The ratio of medium to leaves was 6:1
(w/w). The homogenate was filtered through two layers of Mira-
cloth (Calbiochem). The filtrate constituted the mesophyll cell
fraction. From the residue on the Miracloth, strands of bundle
Although light is required for the assimilation of nitrate into sheath cells were isolated by a combined mechanical maceration-
amino-N (5) and a direct role of light for nitrate reduction has filtration technique described by Chollet and Ogren (6) for corn.
been proposed (33), current evidence indicates that the role of Enzyme Extraction. Whole leaf homogenates were prepared by
light is indirect via generation of reductant and nitrate movement grinding deribbed minced leaf blades with 10 volumes (w/v) of
(4). The concept that light plays a more direct role (via ferredoxin) cold (3 C) medium in a precooled glass TenBroeck homogenizer.
in nitrite assimilation is supported by the work of Magalhaes et al. Separate samples were ground with medium appropriate for each
(22). Klepper et aL ( 19) proposed a general scheme for the relation of the following groups of enzymes: for nitrate reductase (NR),3
of light to nitrate reduction in green leaves. Sugars and/or glyc- NiR, and NAD-GPD, 25 mm K-phosphate (pH 8.6), 5 mm
eraldehyde-3-P formed in the chloroplast, during illumination, Na2EDTA, and 10 mm cysteine; for NADP-glutamate dehydro-
could migrate to the cytoplasm where they could be metabolized genase, the preceding medium was used except for the omission
by the glycolytic enzymes to produce NADH needed for nitrate of EDTA (22); for NAD-malic enzyme and NAD(P)-MDH's, 50
reduction. This scheme was considered applicable to C-4 plants, mM Tris (pH 8.0), 5 mM MgCl2, and 5 mm DTT; Gln Syn, 10 mM
although addition of low concentrations of malate to the in vivo imidazole (pH 8.0), 5 mM MgCl2, 10 mM cysteine, and I mM
assay indicated that malate was not effectively coupled to nitrate Na2EDTA (32).
reduction. It was shown that malate could be coupled to nitrate Enzymes of the mesophyll cell fraction were obtained by grind-
reduction by corn leaves by using higher concentrations (27). ing deribbed leaves as described for.the mesophyll cell fraction,
Investigations with C-4 plants have shown that nitrate assimilation except the media, appropriate for each group of enzymes, was
occurs primarily in the mesophyll cells (I 1, 24, 29); however, these supplemented with 0.33 M sorbitol.
reports did not consider the possibility that the C4 photosynthetic Representative samples of the isolated strands of bundle sheath
system could provide malate as an energy source for nitrate
'Supported by a fellowship grant from MUCIA-Universidad Agraria, Abbreviations: NR: nitrate reductase; NRA; nitrate reductase activity;
LaMolina, Peru to C. A. N., Hatch funds and a Frasch Foundation Grant. NiR: nitrite reductase; NiRA: nitrite reductase activity; Gln Syn: glutamine
This work constitutes a portion of a Ph.D. thesis (C. A. N.) and was synthetase; MDH: malate dehydrogenase; GPD: glyceraldehyde-3-phos-
phate dehydrogenase; DCIP: dichlorophenolindophenol. MES: mesophyll;
presented at the American Society of Plant Physiologists' Meeting in 1974.
2 Present address: Biochemistry and Microbiology Department, Rutgers BS: bundle sheath; FDP: fructose 1,6-diphosphate; PEPase: phosphoenol-
University, P.O. Box 231, New Brunswick, N.J. 08903. pyruvate carboxylase.
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618 Biologists. All rights reserved.
Copyright 1978 American Society of Plant
Plant Physiol. Vol. 62, 1978 NITRATE ASSIMILATION IN CORN LEAVES
strands were ground in the medium appropriate for each group of
enzymes and supplemented with 0.1% Neutronyx 600 (Onyx
Chemical Co., Jersey City, N.J.). The ratio of material to medium
was 1:6 (w/w) and grinding was.done in a precooled glass Ten-
Broeck homogenizer.
Assay Procedures. NRA was assayed as described by Hageman
and Hucklesby (10). The reaction was terminated by adding 0.2
ml of 0.5 M zinc acetate plus 0.2 ml of phenazine methosulfate (46
mg/l) to the 2-ml reaction mixture. After standing 20 min at 25 C,
the extracts were centrifuged at l,000g for 10 min. Aliquots of the
supernatant were used for nitrite determination (9). NiRA was
determined using a dithionite-methylviologen electron donor sys-
tem (15). Gln Syn was determined by the transferase procedure of
Shapiro and Stadtman (32). The other enzymes were measured
spectrophotometrically by the following procedures: NADP-glu-
tamate dehydrogenase (17); NADP-malic enzyme (16); NAD- and
NADP-MDH activities (16); NAD-GPD (8). Nitrate was deter-
mined by the enzymic method of McNamara et al. (23). Chl was
extracted with aqueous acetone 80o (v/v) in the dark. The acetone
extracts were centrifuged at 1,500g for 10 min and the supernatant
used for Chl determination (1). Malate was extracted and assayed
as described by Williamson and Corkey (34). Protein content of
the extracts was determined by the method of Lowry et al. (21)
using BSA fraction V (Nutritional Biochemical Corp.) as standard.
Chloroplast Isolations. Mesophyll and bundle sheath chloro-
plasts were isolated by a modification of the procedure described
by Arntzen (2). The mesophyll fraction suspended (in grinding
medium A) was centrifuged at 2,000g for 10 min and the pellet
resuspended in a suspension medium containing: 0.4 M sorbitol,
20 mm Tricine buffer (pH 7.8), 10 mm KCI, and I mM MgC12.
Bundle sheath cells were broken by hand in a TenBroeck glass
homogenizer and the chloroplasts were obtained after centrifuga-
tion at 10,000g for 20 min. The pellet was resuspended in the same
medium as described for mesophyll chloroplasts.
Photochemical Detenninations. PSII activities (H20-DCIP)
were measured as described by Arntzen (3) in a Hitachi-Perkin
Elmer spectrophotometer modified for direct illumination of the
sample. PSI activities (ascorbate DCIP-methylviologen) were de-
termined by measuring O2 uptake in a Clark electrode. Variable
fluorescence was measured at 685 nm as described by Munday
and Govindjee (25). The slow component of delayed light emission
(DLE) was determined as described by Jursinic and Govindjee
(18) for algae but using much lower Chl concentrations (average
3 Ag/ml).
RESULTS AND DISCUSSION
Characteristics of Mesophyll and Bundle Sheath Fractions. The
characterization of the mesophyll and bundle sheath cell fractions
by microscopic observations, enzymic assays, Chl a/b ratios, and
photochemical activities revealed that the separation techniques
employed effectively separated the fractions. The specific activity
of NADP-MDH in the bundle sheath fraction (8.5 ,umol hr-' mg
Chl-') was only about 5% of the whole leaf activity (141 ,umol
hr-' mg Chl-'). On the other hand, the specific activity of NADP-
malic enzyme in the bundle sheath fraction (911 ,umol hr-' mg
Chl-') was more than three times higher than the whole leaf
activity (258 ,mol hr-1 mg Chl-'). These results are in agreement
with previously reported distribution ratios for these two enzymes
(6). Chl a/b ratios were 5.3 and 3.2 for bundle sheath and
mesophyll chloroplasts, respectively (Table I). Bundle sheath chlo-
roplasts had more (370%) PSI activity (02 evolution) and less
(50%o) PSII activities (DPIP reduction, variable fluorescence, and
delayed light emission) (Table I) than the mesophyll chloroplasts.
Intercellular Distribution of Nitrate and Nitrite Reductase. The
specific activities of NR extractable from the isolated strands of
bundle sheath cells were 5% of those found in the mesophyll cell
fractions (Table II). This is consistent with the concept that the
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Copyright 1978 American Society of Plant Biologists. All rights reserved.
619
Table I. Chlorophyll a/b ratios, photosystem I and
photosystem II activities of mesophyll and bundle sheath
chloroplasts isolated from corn seedling leaves
Mesophyll Bundle Sheath
Chloroplasts Chloroplasts
Chl a/b
System I (pmol 0 hr-l mg Chl-1)
3.2
216
5.3
805
System II (pmol dCIPH2 hr-1 Chl-l)
Fluorescence P-0
118 45
0 3.3 1.6
DLE (slow component) 100 46
Table II. Nitrate and nitrite reductase activities and nitrate
content in mesophyll and bundle sheath fractions isolated
from leaves of corn seedlings
Bundle
Mesophyll Sheath MES/BS
NR (wmol N0- produced hr-1
NiR (imol N0- reduced hr-1
mg-1 protein)
mg-1 protein)
1.34
9.4
0.07
2.10
19.0
4.5
N03 (pmol mg-i protein) 7.3 0.46 16.0
bulk of the nitrate assimilation occurs in the mesophyll cells.
Similar results were obtained with several other corn hybrids (the
average specific activity of NR extracted from strands of bundle
sheath cells was 0.08). While it can be argued that the NR is a
constituent of the bundle sheath cells, it is more plausible that the
trace of NR is due to mesophyll contamination. This is supported
by the observation that NADP-MDH has a similar distribution
between mesophyll and bundle sheath cell fractions (i.e. ratio of
19:1, MES/BS).
The distribution of nitrate concentration between mesophyll
and bundle sheath fractions was comparable (ratio of 16:1,
MES/BS) to that of NR. This ratio may not reflect the in situ
distribution because aqueous separation techniques were used to
isolate the bundle sheath strands. If the ratio is a valid estimation
of distribution it would imply a preferential movement of nitrate
from the xylem to the mesophyll cells. Regardless of the distri-
bution of nitrate, the bundle sheath cells may lack the ability to
induce NR. All attempts to induce NR in isolated strands of
bundle sheath cells by techniques (light, N03) that will induce
NR in sections of corn leaf blades were unsuccessful.
Although the specific activity of NiR of the mesophyll fraction
is 4.5-fold higher than that of the bundle sheath cells, the amount
of NiR in the bundle sheath cells indicates that it is a constituent
enzyme. This raises a question as to the physiological role of NiR
in the bundle sheath cells. Possible explanations are: (a) a small
amount of nitrate is assimilated in the bundle sheath cells; (b) it
is present to assimilate nitrite that is transferred from the meso-
phyll cells; and (c) conditions in the bundle sheath cell permit the
induction of NiR and it has no physiological role.
The absence of NRA and lower amounts of NiRA and PSII in
the bundle sheath cells (Table I and ref. 26) support the contention
that the bulk of the nitrate is assimilated in the mesophyll cells.
NiR extracted from chloroplasts isolated from the mesophyll
supernatant had higher activity (7.8 ,umol N02 reduced hr-' mg-'
protein) than NiRA of the mesophyll supernatant (1.8 ,umol).
These findings are consistent with the reports that NiR is localized
in the chloroplasts (22, 26, 30).
Nitrate Reduction and Malate Metabolism. NRA and malate
concentration, in corn leaf blades, show similar diurnal variations
(Fig. 1). While the increases in NRA and malate concentration
were similar during both illumination periods, malate concentra-
tion decreased more rapidly than NRA in the dark period. Because
light is required for the synthesis of NRA in green leaves, the
decrease in NRA during the dark period is attributed to the
continued degradation of the enzyme. However, the light effect is
probably indirect because induction of NRA was also observed in
the roots of the same plants upon addition of nitrate (Fig. 1, inset).
These data show no obligatory relationship between nitrate re-
duction and malate concentration in the mesophyll cell fraction.
Canvin and Atkins (5) have shown that nitrate is not assimilated
under dark anaerobic conditions; therefore, reactions other than
620
nitrate reduction are responsible for the decrease in malate con-
centration.
These data (Fig. I) show that the rate of malate production
during illumination is sufficiently high to permit accumulation in
the mesophyll cell fraction. Some of the malate could be oxidized
to provide energy for nitrate assimilation in the mesophyll cells.
Extracts from mesophyll cells contained NAD-MDH in levels
adequate to support in situ levels of nitrate assimilation. Using the
coupled assay (NAD-MDH-NR), nitrate reduction of mesophyll
extracts fortified with 20 mm malate and 0.4 mm NAD was 80%
that obtained with comparable aliquots of extract supplied with
0.4 mm NADH (control activity, 0.8 ,tmol NO2 hr-' mg-' protein)
as electron donor for NR. The best estimates of in vivo NRA in
corn leaf blades are lower than the 0.64 ,tmol NO2- hr-' mg-1
protein rates observed here. The coupling of photosynthesis to
nitrate reduction could operate in situ via the proposed malate-
oxaloacetate shuttle that generates NADH in the cytoplasm (13,
14).
Intercellular Distribution of NADH-generating Enzymes. Ad-
dition of malate (27) or glyceraldehyde-3-P (19) to the in vivo NR
assay medium usually increases the rate of nitrate reduction.
Presumably this increase is due to the uptake and oxidation of the
metabolites by the leaf segments, thereby increasing the availabil-
ity of NADH for nitrate reduction. Intercellular distribution of
NAD-MD and NAD-GPD was determined to verify if the local-
ization of these enzymes was consistent with this view.
Data of Table III show that these two enzymes are equally
distributed between mesophyll and bundle sheath cell. Because
NR, NAD-GPD, and at least a portion of NAD-MD are located
in the cytoplasm of the mesophyll cells (19, 30, 31), it is possible
for either malate or glyceraldehyde-3-P to be coupled to nitrate
reduction by the appropriate enzymes.
Interceflular Distribution of Glutamine Synthetase and
NADPH-Glutamate Dehydrogenase. The distribution patterns of
these two enzymes, known to be associated with the primary
incorporation of ammonia into amino acids (20, 22), were deter-
mined. A higher concentration of NADPH-glutamate dehydro-
genase was found in the bundle sheath cell fraction than in the
whole leaf fraction (1.5-fold protein basis, 2.3-fold Chl basis,
18-
16-
3 14-
(0 12-
0)
z 10-
8-
E 6-
:4 4-
cr 2-
z
8am 8pm Sam 2pm
FIG. 1. Induction of NRA and malate content in the leaves of intact
B14 x B37 corn seedlings. Seedlings were grown in liquid cultures con-
taining traces of nitrate. Induction was initiated by adding nitrate (10 mM)
to the solutions at 8 AM on day 1. Inset: NRA in roots of same plants.
Table III. Intercellular distribution of NAD-malate
dehydrogenase and NAD-glyceraldehyde
phosphate dehydrogenase
Leaf samples were obtained from 12-day-old XL-81
corn seedlings cultured in Vermiculite with full
strength Hoagland solution.
Enzyme Activities
Leaf fraction NAD-MDH NAD-GPD
(pmol hr-l mg protein-l)
Whole leaf 197 15
Mesophyll 180 19
Bundle sheath 158 19
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Copyright 1978 American Society of Plant Biologists. All rights reserved.
NEYRA AND HAGEMAN Plant Physiol. Vol. 62, 1978
Table IV). In contrast to NADPH-glutamate dehydrogenase, the
reverse ratio of Gln Syn in bundle sheath and whole leaf fractions
(Table IV) shows that relatively high levels of Gln Syn occur in
both bundle sheath and mesophyll cells. This observation made
and reported in 1974 has recently been confirmed by other workers
(I 1, 29). The existence of Gln Syn in the mesophyll cells provides
a feasible metabolic system for incorporation of the ammonia
produced by NO2 reduction in these cells. Such a system avoids
the need for a flow of free ammonium ions from the mesophyll to
the bundle sheath cells (24).
Several lines of evidence indicate that Gln Syn may play a
major role in the primary incorporation of ammonia into amino
acids in corn leaves: (a) it is present in the mesophyll cells, which
is the major site of nitrate and nitrite reduction; (b) the specific
activity of Gln Syn is severalfold that of NR and comparable to
NiR (cf. Tables II and IV), thereby able to accommodate the in
vivo rates of nitrate reduction; (c) it has been shown to be located
in chloroplasts (28) known to be the major site of de novo
formation of amino acids (22); and (d) Gln Syn has a relatively
low Km for ammonia in comparison with glutamate dehydrogenase
(20).
Proposed Pathway for Nitrate Assimilation in Corn Leaves.
The sequence of reactions of Figure 2 illustrates the concept that
the mesophyll cells are the major site for nitrate assimilation in
corn leaves. This concept is based on the following observations:
(a) the cellular distribution of enzymes associated with nitrate
assimilation; (b) the availability of nitrate and energy sources for
nitrate reduction; (c) the flow of metabolites associated with the
C-4 pathway of photosynthesis (7, 12); and (d) the operation of a
malate-oxaloacetate shuttle proposed to operate in the transfer of
reducing equivalents from the chloroplast to the cytoplasm (13,
14). Photosynthetically produced malate (mesophyll chloroplast)
and/or sugars (bundle sheath chloroplasts) are the most logical
sources of energy for nitrate reduction which appears to be local-
ized in the cytoplasm of the mesophyll cells. Oxidation of malate
and/or glyceraldehyde-3-P could provide NADH for nitrate re-
duction and other NADH-requiring reactions. Data of Figure 3
show that fructose-diP or malate can be used by leaf segments
under in vivo assay conditions to enhance nitrate reduction. Al-
though these results are plausible and logical, they do not prove
that these two metabolites are actually coupled to nitrate reduction
in the intact plant. In the chloroplasts of the mesophyll cells,
I03. Table IV. Glutamate dehydrogenase (NADPH) and
0
glutamine synthetase activities in whole
12 L leaves and bundle sheath extracts
Enzyme Activities
10 o Leaf Fraction NADPH-GDH Gln Syn
8 E (imol hr-I mg protein-1)
12.1 (125)
Whole leaf 0.65 (6.7)*
6 4 Bundle sheath 0.95 (15.4) 9.1 (146)
-441!a *Activity values in parentheses are per Ug Chl.
2 -i
CELLS
MESOPHYLL CELLS
RFd
N
CHLOROPLAST
N
1 BUNDLE SHEATH
CHLOROPLAST
/No;-14NH;-.-RNHn
FIG. 2. Proposed pathway for nitrate assimilation in corn leaves.
Plant Physiol. Vol. 62, 1978 NITRATE ASSIMILATION IN CORN LEAVES
8 -
7- MALATE +NAD
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FIG. 3. Effect of FDP, malate, and NAD on in vivo nitrate reduction
by corn leaf sections. Samples were obtained from the third leaf of 11-
day-old seedlings (XL-8 1) and vacuum-infiltrated with a medium contain-
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propanol. Additions in t,mol/ml were: FDP, 5; malate, 20; NAD, 3.
nitrite is reduced to ammonia by NiR using ferredoxin as the
electron donor (26). Reduced ferredoxin is generated by light
energy via the electron transport system of the chloroplasts. Am-
monia can be primarily incorporated into glutamine via Gln Syn
in the mesophyll chloroplasts.
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