Planta (1994)194:279-286 P l a n t a

�9 Springer-Verlag 1994

In-situ localization of nitrate reductase in

Elena Fedorova*, John S. Greenwood, Ann Oaks

Department of Botany, University of Guelph, Guelph, ON, N1G 2W1, Canada

Received: 22 October 1993 / Accepted: 29 November 1993

maize roots

Abstract. The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues of Z e a m a y s L. (W64A 'W182L) was determined using post-embedding im- munogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cyto- plasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchy- mal cells of the pith lying next to the xylem. A positive signal for N R protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when afffinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant N R protein when adequate controls and signal-enhance- ment procedures were used.

Key words: Nitrate reductase (immunolocalization) - Nitrogen assimilation (roots) - Z e a (N assimilation)

Introduction

Nitrate assimilation is a highly regulated process that involves the t ransport of nitrate into the cell, its subse- quent reduction to nitrite and ammonium, and the incor- porat ion of ammonium into carbon skeletons (Oaks and Hirel 1985). Nitrate reductase (NR) is the first enzyme involved in the actual assimilation of nitrate in plants. It catalyzes the conversion of nitrate to nitrite, using either N A D H (green leaves, EC 1.6.6.1) or NAD(P)H (roots, EC 1.6.6.2) as the electron donor.

* Present address: K.A. Timiryasev Institute of Plant Physiology, Moscow, Russia Abbreviations: IgG = immunoglobulin G; NR = nitrate reduc- tase; PEPCase = phosphoenolpyruvate carboxylase Correspondence to: A. Oaks; FAX: 1 (519) 767 1991

Nitrate reductase is both substrate- and light-in- ducible in green leaves. In roots, N O 3 is impor tant for the induction of NR, but the light effect is not pro- nounced (Bowsher et al. 1991). Glucose additions can also result in enhanced levels of N R activity in both leaf and root tisues. According to results obtained using cell- fractionation and biochemical techniques, N R should be located in the cytosol in cells of both roots and leaves (Dalling et al. 1972a, b). Immunocytochemical tech- niques, using antibodies prepared against purified NR, should provide superior evidence for the cellular location of the N R protein. However, because N R is a minor protein and because it has been difficult to purify to ho- mogeneity (Sechley et al. 1992; Redinbaugh and Camp- bell 1983), this technique is frought with problems. With different antibodies or different experimental systems the enzyme has been localized in the plasma membrane in barley roots (Ward et al. 1988), in the cytoplasm and in the chloroplasts of spinach leaf cells (Kamachi et al. 1987; Roldan et al. 1987), and in the cytosol of maize-leaf mes- ophyll cells (Vaughn and Campbell 1988). In this paper we describe for the first time the distribution and local- ization of N R in cells of root tissues of Z e a mays, as determined by immunocytochemistry.

Materials and methods

Plant material. Seeds of Zea mays L. (W64A x W182E) were pur- chased from Wisconsin Seed Foundation (Madison, Wis., USA). Seeds were sown on 0.9% agar made with 1/10-strength Hoagland's solution containing no nitrate (Aslam and Oaks 1976). After 2 d, the seedlings were transferred to an aerated hydroponic system which contained 1/10 Hoagland solution. Potassium nitrate was added at 0.1, 0.5, 1, 5, or 10mM, as required, 24 h prior to collection. Seedlings were incubated under a 16-h day (light level of 225 mmol photons.m~.s 1), 8-h night regime at a constant 29~

Assay of NR activity. Roots and shoots were collected separately, frozen in liquid N 2 and ground to a fine powder. Extracts of these powders were used for the analysis of NR activity following the method of Long and Oaks (1990). One gram of frozen powder was ground with 4 mL of extraction buffer (25 mM Tris-HC1, pH 8.5;

280 E. Fedorova et al.: Nitrate-reductase localization in corn roots

1 mM EDTA; 20 mM FAD; 1% [w/v] bovine serum albumin; 1 mM dithiothreitol; 10 mM cysteine; 10 mM chymostatin dis- solved in dimethylsufoxide). The extracts were centrifuged at 10 000.g, filtered through Miracloth (Calbiochem, La Jolla, Calif., USA) and added to an assay mixture consisting of 0.2 mL Hepes buffer (0.65 M, pH 7.2), 0.2 ml KNO3 (0.1 M) and 0.1 ml extract in a total volume of 0.5 ml. The reaction was performed according to Long and Oaks (1990). Briefly, water was added to bring the reac- tion mixture to 1.4 ml. The reaction was started by the addition of 0.1 ml N A D H (3.6 mg.ml 1 in 0.04 M KPO4, pH 7.0) and the reac- tion mixture incubated at 28~ for 20 min. Addition of 0.1 ml alco- hol dehydrogenase (0.5 mg.ml 1 0.1 M KPO4, pH 7.0) and 0.1 ml 2% (v/v) acetaldehyde stopped the reaction. Nitrite produced was determined by measuring A54 o 30 min following the addition of 1 ml 1% (w/v) sulfanilamide in 1 N HC1 and 1 ml 0.01% (w/v) aqueous N-l-naphthyalenediamine dihydrochloride to the assay mixture.

Gel electrophoresis and immunoblotting procedures. Tissues were ex- tracted as above, except that bovine serum albumin was omitted from the extraction solution. Analysis by SDS-PAGE was per- formed according to Poulle et al. (1987)with 7.5% (w/v) polyacry- lamide gels using the Biorad (Biorad Laboratories Ltd., Missis- sauga, Ontario, Canada) MiniGel apparatus and loading 10 ml of extract per lane. Immunoblotting was performed according to Tow- bin et al. (1979) onto nitrocellulose (BioBlot-NC; Costar, Cam- bridge, Mass., USA) at 100 V for 1 h, using the Biorad Mini trans- blot apparatus. The nitrocellulose was blocked overnight with 20 mM Tris-HC1, 500 mM NaC1 containing 0.5% Tween 20 (TTBS). Anti-NR antibody was diluted 1:400 (3.5 mg.ml ~) with TTBS and the nitrocellulose was incubated in this solution for 3-4 h. The nitrocellulose was washed with TTBS two times for 15 min, and the reacted protein was visualized with a goat-anti-rabbit immunoglob- ulin G (IgG) alkaline-phosphatase conjugate (BioRad), as described previously (Goodfellow and Oaks 1993).

Preparation and purification of the anti-NR antibody. Maize leaf NR was chromatographically purified over Blue Sepharose according to Poulle et al. (1987). After the Blue Sepharose affinity step, NR was purified by passage over a diethylaminoethyl-cellulose column followed by native gel electrophoresis and electroelution (Long and Oaks 1990). Purified protein was injected into New Zealand white rabbits (200 mg protein.ml i Freund's incomplete adjuvant per in- jection, four weekly injections). The antiserum was precipitated by with (NH4)2SO 4 (45% saturation). After centrifugation at 10 000.g for 15 min, the pellet was washed with 1.75 M (NH4)2SO 4. The pellet was resuspended in a volume of 10 mM KPO4 buffer (pH 8.0) equiv- alent to the initial volume of the crude antiserum, and dialyzed overnight against the same buffer (Poulle et al. 1987; Long and Oaks 1990).

Both the crude antiserum and IgG fractions reacted to more than just NR on Western blots. To reduce non-specific reactions, antibodies were subsequently purified according to the method of Smith and Fisher (1984), using nitrocellulose blots. Regions con- taining NR bands were excised from the blot and each fragment was then eluted 3 x 30 s with 5 mM glycine-HC1 (pH 2.3), 500 mM NaC1, 0.5% (v/v) Tween-20. The pH was adjusted to neutrality us- ing NaPO4. Antibodies obtained after these procedures were pres- sure-concentrated, using an Amicon Centricon 100 membrane (Amicon Corp., Danvers, Mass., USA). The resultant 30-fold-con- centrated (52 mg.ml 1) solution of the antibody was used for im- munolabelling. Antibodies were also competed with purified phos- phoenolpyruvate carboxylase (PEPCase), a major protein in maize leaves which has a subunit of approximately the same apparent molecular weight as NR (100 vs. 116 kDa, respectively).

Cell fractionation. Isolation of plastids from corn roots was per- formed according the method of Emes and England (1986), with the exception that buffers did not contain bovine serum albumin. Sliced roots were homogenized in 50 mM N-[2-hydroxy-l,l-bis(hydrox- ymethyl)ethyl]glycine (Tricine)-NaOH buffer (pH 7.9) containing 0.33 M sorbitol, 1 mM EDTA, 1 mM MgC12 (buffer A). Material

was filtred through six layers of muslin and centrifuged at 200"g for 5 min. Aliquots of supernatants (5 ml) were underlaid with 10 ml buffer A containing 10% Percoll (Pharmacia P-L Biochemicals Inc., Milwaukee, Wis., USA) and centrifuged at 4000.g for 10 min. The resulting pellet was homogenized in a mortar and pestle in 100 mM Tris-HC1 (pH 7.5), 1 mM EDTA, and 10 mM 2-mercaptoethanol. The homogenate was centrifuged at 12 000.g to remove the mem- brane fraction, and the supernatant proteins were analyzed by SDS- PAGE and Western blotting.

Isolation of the chloroplasts was performed according to Wu and Wedding (1985). Sliced leaves were incubated in 0.1 M Hepes buffer (pH 6.2) containing 0.33 M mannitol, 1 mM dithiothreitol, with 1% cellulase and 0.2% pectinase, for 3 h. The slurry was fil- tered through one layer of cheesecloth and centrifuged at 100.g for 2 min. The pellet was resuspended in the same buffer, loaded onto 10% Percoll, and centrifuged for 10 min at 100.g. Protoplasts float- ed on the Percoll were collected, ground in a mortar, and layered on a 40% Percoll solution. Following centrifugation at 3300.g for 2 min, the sedimented chloroplasts were collected as intact chloro- plasts. Chloroplasts suspended in the 40% layer were collected, sedimented at 12 000.g for 10 min, and the pellet disrupted ultra- sonically. Proteins from the chloroplast-enriched fractions were subjected to SDS-PAGE and Western blotting.

Electron microscopy. For electron microscopy, root tissues were col- lected after 6 d (3 d after transfer to hydroponic conditions, 24 h after induction of NR) and fixed in 4% depolymerized paraformaldehyde, 0.3% glutaraldehyde in 0.05 mM potassium- phosphate buffer with added 0.25 M sucrose (pH 7.3), for 16 h at 4~ The fixed tissues were then rinsed in the same buffer three times for 30 min, dehydrated through a graded ethanol series, and infil- trated with Lowicryl K4 M resin (J.B. EM Services Inc., Montreal, Quebec, Canada) at -20~ Polymerization was performed in gelatin capsules under UV light at -20~ Sections, 70-100 nm thick, were obtained using an LKB Nova ultramicrotome (LKB, Upsala, Sweden) with glass knives. Sections were mounted on 50- mesh gold grids and processed for immunolocalization of NR prior to observation using a JEOL 100 CX scanning-transmission elec- tron microscope (JEOL, Tokyo, Japan).

Immunolabeling. Post-embedding immunolabeling of NR was per- formed according to protocols modifed from Greenwood and Chrispeels (1985a). Briefly, grids were incubated in 0.01 M KPO4, 0.15 M NaCI (pH 7.4) with 0.5% Tween 20 (PBST) and 0.5% teleost gelatin as a blocking agent for 15 min; incubated in anti-NR anti- body in PBST, 2.5 h to overnight at 4~ washed through six changes of PBST plus gelatin, 10 min each; exposed to 12-nm col- loidal gold, prepared according to Slot and Geuze (1985), conjugat- ed to affinity purified goat-anti-rabbit IgG (Sigma Chemical Co., St. Louis, Mo., USA) and diluted to A525 = 0.4 in PBST with gelatin, 2.5 h at 4~ washed through six changes of PBST, 10 min each and two changes of distilled H20, 5 min each. Grids were then dried and stained using 2% uranyl acetate in distilled H20, 20 min at 60~ followed by Reynold's lead citrate, 1 min at 20~ In the case of root tissues, 12-nm colloidal gold conjugated to affinity-purified donkey anti-goat IgG (Sigma) was used as a tertiary label to enhance the signal.

Localization of NR was also performed at the light level using silver enhancement of colloidal-gold labeling, following the proto- col of Wetzel and Greenwood (1991).

Results

Induction of NR. In the p r e l i m i n a r y e x p e r i m e n t s , N R was i n d u c e d by e x p o s i n g 6 -d -o ld seed l ings to 0, 0.5, 1, 5 a n d 10 m M K N O 3 for 24 h. T h e N R ac t iv i ty in b o t h s h o o t s a n d in r o o t s i n c r e a s e d wi th i nc r ea s ing c o n c e n t r a t i o n o f NO3- (Fig. 1). In the i m m u n o b l o t s , the a n t i - N R a n t i b o d y

E. Fedorova et al.: Nitrate-reductase localization in corn roots

Nitrate reductase activity

1 ROOT ~ SHOOT

"6 E :3-

-V

- 0 m M 0 . 5 m M 1 mM 5 m M 1 0 m M

NITRATE IN GROWTH MEDIUM

Fig. 1. Nitrate reductase activity (mmol nitrite.g a.h 1) in the shoots and the roots of maize plants exposed to different concentrations of nitrate for 24 h

preparation detected a number of proteins (Fig. 2). How- ever, by comparing the crude extracts to the immunoblot obtained with a partially purified NR protein (lane 6, Fig. 2), it is evident that the NR-protein content also in- creased with increasing levels of N O r used (Fig. 2).

According to the results obtained by Kamachi et al. (1987) and Roldan et al. (1987), N R protein may be locat- ed in the chloroplasts. In preliminary experiments using maize leaf tissue and fractionated ant i-NR IgG we were able to establish that most of the N R protein was in the cytosol of mesophyll cells, in agreement with the results of Vaughn and Campbell (1988). There was no cytosolic signal when the plants were grown in the absence of NO 3- or when the antibody was competed with purified N R protein (data not shown). Competition with purified PEPCase protein had no effect on the N R signal (data

281

not shown). However, as with the results of other investi- gators, in particular Kamachi et al. (1987) and Roldan et al. (1987), a signal for N R protein could be seen in chloro- plasts in our leaf tissue sections and in the cell wall-plas- malemma interface. To determine whether this was, in fact, N R protein, chloroplasts were isolated and imm- noblot analysis performed using NR-specific antibodies. The immunoblot (Fig. 3) illustrates that some plastid- specific proteins of Mr 69 kDa are recognized by the frac- tionated anti-NR IgG, but that 106-kDa N R protein is present in neither leaf chloroplast nor root plastid frac- tions. Some cross-reacting proteins are also present in the cytosolic fraction.

Purification of anti-NR antibody provides higher specifici- ty. In order to alleviate possible cross-reactivity with non-NR proteins in the root tissue sections, the crude antibody preparation was purified using an affinity pro- cedure (Smith and Fisher 1984). A Western blot (Fig. 4) was prepared using total protein extracts from 6-d-old shoots induced for 24 h, and using anti-NR IgG obtained after purification. It is evident that the purification of the anti-NR IgG using the procedures of Smith and Fisher (1984) provides a much more specific antibody prepara- tion. The 116-kDa NR protein is readily identified in the total protein extracts with very little to no cross-reactivi- ty with other extractable proteins. Proteins from either the cytosol or the plastids that gave cross-reactivity with the crude antiserum are no longer detected. Antibodies purified in this way were used for subsequent electron- microscopic and light-level immunolabelling.

Immunolocalization of N R in root tissues. Plants exposed for 24 h to 10 mM NO3, and to zero N O r as a control, were used to examine the immunolocalization of NR in the maize root tissues. Figure 5A illustrates that the frac- tionated anti-NR IgG recognized sites or proteins

Fig. 2. Western-blot analysis, using anti-NR antibody, of proteins from crude extracts of maize plants. Lanes 1-5 represent extracts from shoots of plants exposed to different concentrations of nitrate for 24 h. Lane 1, 10 mM nitrate; lane 2, 5 mM; lane 3, 1 mM; lane 4, 0.5 mM; lane 5, 0 mM (no nitrate). Lane 6, partially purified NR

from leaves. Lanes 7-11 illustrate extracts from roots of plants ex- posed to different concentrations of nitrate for 24 h. Lane 7, no nitrate; lane 8, 0.5 mM nitrate; lane 9, 1 mM nitrate; lane 10, 5 mM nitrate; lane 11, 10 mM nitrate. Arrowhead indicates the position of authentic purified NR run simultaneously

282 E. Fedorova et al.: Nitrate-reductase localization in corn roots

Fig. 3. Western-blot analysis using anti-NR antibody, of proteins from cytosolic and plastid fractions following exposure of maize plants to 10mM nitrate. Lane 1, leaf chloroplast fraction from plants exposed to 10 mM nitrate. Lane 2, leaf chloroplast fraction from zero-nitrate control plants. Lane 3, leaf cytosolic fraction from plants exposed to 10 mM nitrate. Lane 4, leaf cytosolic fraction

from zero-nitrate control plants. Lane 5, plastid fraction from roots of plants exposed to 10 mM nitrate. Lane 6, plastid fraction from roots of zero-nitrate controls. Lane 7, cytoplasmic fraction from roots of plants exposed to 10 mM nitrate. Lane 8, cytoplasmic frac- tion from roots of zero-nitrate controls. Arrowhead, position of authentic NR run simultaneously

Fig. 4. Western-blot analysis of proteins from shoot extracts using affinity-purified anti-NR antibody. Lane 1, crude total protein extract from zero-nitrate control plants. Lane 2, crude total protein ex- tracts from plants exposed to 10 mM ni- trate. Note that the specificity of the anti- NR antibody preparation for NR has been improved by purification. Arrow- head indicates the position of authentic purified NR run simultaneously

specific to the cell wall and cell wall-plasmalemma inter- face as well as proteins in the cytoplasm.

It is known that the PEPCase polypeptide has almost the same apparent molecular weight as the N R subunit and one of the major problems with N R preparations used for ant ibody product ion is that they can be contam- inated with PEPCase. This is particularly true when the antibodies are prepared using extracts from leaves of C4 plants, such as maize, where PEPCase can account for 10-20% of the total protein (Sechley et al. 1992). Phos- phoenolpyruvate carboxylase is known to be cytoplasmi- cally localized in the mesophyll cells of C4 plant leaves, but is not found in the bundle-sheath cells (Ueno 1992). Vaughn and Campbell (1988) obtained a distribution of N R protein, as determined by immunolocalization, that was identical to the distribution of PEPCase found by Ueno (1992) in leaves of another C4 species. This distribu- tion would be predicted on the basis of biochemical evi- dence. However, because of the possibility of contamina- tion with PEPCase (Solomonson and Barber 1990; Sech- ley et al. 1992), the N R antibodies used in our localiza- tion experiments were purified by the methods of Smith

and Fisher (1984) and then competed with PEPCase protein at a concentration of 77 mg.ml 1 prior to use for immunolocalization. Finally, because N R protein repre- sents only 0.01 0.05% of the total extractable protein found in NO3-induced plant cells (Redinbaugh and Campbell 1983), a double-bridge immunolocalization procedure (12-nm-colloidal-gold-tagged goat anti-rabbit IgG followed by 12-nm-colloidal-gold-tagged donkey anti-goat IgG) was used to enhance the N R signal.

The N R protein was localized in the cytoplasm of the root cells using the purified ant i -NR antibodies chal- lenged with PEPCase (Fig. 5B-E). Some labeling of the cell wall-plasmalemma region was observed (Fig. 5C-E); however, a clustered arrangement of gold particles, rather than individual particles, would be more consis- tent with the localization of N R due to the multiplicity of the signal obtained with the double-bridge procedure. Clusters of particles were rarely observed in association with the cell wall-plasmalemma region (Fig. 5B D).

Distribution of N R protein across the root was fol- lowed using silver-enhanced immunogold labeling at the light-microscopy level. Sections of roots which had been exposed to NO3 were made from the mature regions (3 cm from the tip). Figure 6A, C illustrates sections of roots of plants exposed to 10 mM NO 3 . In the sections from mature regions of the root, note that the silver grains, indicating the location of N R in the tissues, are arranged in rows along both sides of the cell wall (Fig. 6C). This is the expected pattern of labeling for a cytoplasmically located antigen. When seedlings were ex- posed to nitrate, labeling of both the cortical and epider- mal cells was qualitatively the same (Fig. 6A, C). Nitrate reductase is localized primarily in the parenchymal cells of the cortex, and in the cells of the pericycle and par- enchyma within the vascular cylinder. A weaker signal was obtained in parenchymal cells of the pith lying next to the xylem and in cells of the endodermis.

Labeling of all tissues in the zero-NO3 control was much reduced compared to that of the NO3-exposed

Fig. 5A-E. Immunolabeling of the root tissue from plants exposed to 10 mM nitrate. A Immunolabeling using fractionated, unpurified anti-NR IgG. Colloidal gold particles are present over the ce[I wall- plasmalemma region, and to a lesser extent over the cytosol (arrow- heads). '26 000. B-E Localization of NR in root tissue, following affinity purification of the antibody and challenging the antibody with PEPCase. The signal is present in the cytoplasm. B '36 800, C

%0 000, D '30 000, E '40 000. Gold particles, indicating the location of NR, are distributed in clusters in the cytoplasm of cortical (B, C, E) and pericycle (D) cells (arrowheads). The clustering of the gold particles is due to the double-bridge immunolabeling procedure used, enhancing the NR signal. Note the absence of cell wall label- ing in preparations treated with the purified antibody. C W, cell wall; M, mitochondrion; P1, plastid. Bars = 0.25 mm

Fig. 6A-D. Silver-enhanced immunogold localization of NR in roots, using affinity-purified ant i -NR antibodies and challenging with purified PEPCase. A, C Cross-sections of roots exposed to 10 mM nitrate. B, D Cross-sections of roots from zero-nitrate con- trols. Arrowheads indicate silver grains. Note that the silver grains, indicating the location of NR in the tissues, are arranged in rows

along both sides of the cell wall. Labeling is seen in the epidermal and cortical cells (E, C, respectively), cells of the pericycle (P) and in the parenchymal cells surrounding the vasculature. Labeling is re- duced in cells of the endodermis (En) and in the pith (Pt). Differen- tial interference contrast. Bars = 200 mm (A, B), and 25 mm (C, D)

E. Fedorova et al.: Nitrate-reductase localization in corn roots 285

Fig. 7A, B. Immunolabeling controls. A Immunolabelling of the root tissue from zero-nitrate control plants. Note lack of labeling (clusters of black dots as are seen in Fig. 5) in the cytoplasm of

zero-nitrate control roots, although a minor signal is seen over the cell walls. '40 000. B Preimmune fractionated IgG control. '20 000, N, nucleus. Bars = 0.25 mm

tissue and no clustered signal, indicative of NR location, was observed (Fig. 6B, D and 7A). Labeling using preim- mune serum was not associated with any particular cellu- lar compartment, and was very sparse (Fig. 7B). Thus, in maize roots, N R appears to be a cytosolic protein. Ni- trate-reductase protein is present in the cytoplasm of the parechymal cells throughout the root, with the possible exception of the endodermal cells.

Discussion

As expected, increasing the concentration of NO 3 result- ed in increased levels of NR activity and protein in both the root and shoot tissue (Figs. 1, 2). By comparing the NO3--treated and zero-NO 3 controls we have demon- strated that N R in treated maize root tissues is located in the cytoplasm, supporting previous biochemical studies (Dalling et al. 1972b; Oaks and Gadal 1979). According to our results, an N R protein with a polypeptide size of 106 kDa is present in neither the chloroplast fraction from the leaves, nor in the plastid fraction of corn roots (Fig. 3). Polyclonal anti-NR antibodies react with some proteins in the plastid fractions, but the proteins do not migrate to the same position as authentic N R in SDS- polyacrylamide gels, and the signal is lost when affinity- purified antibody is used (Fig. 4). Thus, the labeling of chloroplasts obtained by Kamachi et al. (1987) may have

been background since polyclonal anti-NR sera com- monly recognize non-NR proteins, some of which may be located in the plastid.

Our results demonstrate that sections of Lowicryl-em- bedded tissue are adequate for the post-embedding local- ization of non-abundant proteins, such as NR, providing that adequate controls and signal-enhancement proce- dures are used. The pre-enabedding immunolocalization procedures of Vaughan and Campbell (1988), as well as labeling of ultrathin cryosections followed by on-grid embedding (Greenwood et al. 1984; Greenwood and Chrispeels 1985b), are superior methods for such local- izations but are technically far more difficult. In our preparations, NR protein appears to be freely distributed in the cytoplasm of the root cells. Nitrate-reductase protein has also been localized in the cytosol of maize leaf mesophyll cells using a pre-embedding immunolabeling procedure (Vaughn and Campbell 1988). The NR protein was not obviously localized to the plasmalemma in maize root cells, as has been suggested with barley (Ward et al. 1988). However, it may well be that our detection method is not sensitive enough to clearly establish the presence of NR in the membrane (5% of the cytosolic signal accord- ing to Ward et al. 1988). Since the plasmalemma signal could be related to the NR protein or to a NO3--specific permease (Ward et al. 1988), the appropriate material to use to clarify this point would be the barley mutants described by Warner and Huffaker (1989) which lack the

286 E. Fedorova et al.: Nitrate-reductase localization in corn roots

p ro t e in for the N A D H (specific)- and N A D ( P ) H (bis- pecif ic)-NRs, yet a p p a r e n t l y have a n o r m a l up t ake sys- tem.

A c c o r d i n g to ou r results, N R p ro t e in was present in high levels in the e p i d e r m a l and cor t ica l cells, and in p a r e n c h y m a l a n d per icycle cells of the centra l cylinder. E n d o d e r m a l cells were no t l abe led and p a r e n c h y m a l cells of the p i th were only weak ly labeled. Our results do no t comple t e ly agree wi th the mic rosurg ica l d a t a ob t a ined by Rufty et al. (1986), where N R was found in all d issected regions of roo t s exposed to 20 m M N O 3 , but a p p e a r e d to be c o n c e n t r a t e d in the ep ide rma l cells. The differences m a y be exp la ined by differences in exper imenta l t rea t - men t s given the p lan t s p r io r to analysis . In tac t p lan ts were fed with n i t ra te in ou r exper iments . Rufty et al. (1986) r emoved the mesoco ty l s p r io r to the 20 h d a r k t r ea tmen t wi th n i t ra te and covered the cut shoo t bases wi th d ry co t t on to a b s o r b exudate . These cond i t ions p r o b a b l y res t r ic ted n o r m a l t r ansp i r a t ion , c a rbon flow and exchange be tween the source and sink of the seedling. As a result , the express ion of N R pro te in within the roo t was p r o b a b l y al tered.

O u r results show tha t N R p ro t e in is found in the cyto- p l a s m of e p i d e r m a l and cor t ica l cells, and in cells of per i- cycle and p a r e n c h y m a s u r r o u n d i n g the vascu la r tissue in the stele of maize roots . In add i t ion , N R in maize roo t s is p resen t in high enough levels to accoun t for a subs tan t ia l r educ t ion of n i t ra te in the roo t s (50% of tha t seen in leaf t issues; Fig. 1). Thus ma ize roo t s cou ld have an impor - tan t role in the overa l l m e t a b o l i s m of ni t rate . Our obser- va t ions s u p p o r t the sugges t ion of G o j a n et al. (1986) tha t roo t s cou ld accoun t for ~ p p r o x i m a t e l y one - th i rd of the whole p l an t r educ t ion of NO3 in Zea mays.

This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F..

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