Nitrate-Induced Changes in Protein Synthesis and Translation ... - NCBI

9 downloads 82 Views 2MB Size Report
Dec 10, 1986 - PW Ludden, JE Burris, eds, Nitrogen Fixation and CO2 Metabolism. Else- vier, Amsterdam ... John Wiley & Sons, New York. 26. SOMERS DA ...
Plant Physiol. (1987) 84, 52-57 0032-0889/87/84/0052/06/$0 1.00/0

Nitrate-Induced Changes in Protein Synthesis and Translation of RNA in Maize Roots' Received for publication August 13, 1986 and in revised form December 10, 1986

PETER R. MCCLURE*, THOMAS E. OMHOLT, GARY M. PACE, AND PIERRE-YVES BOUTHYETTE Section of Plant Biology, Cornell University, Ithaca, New York (P.R.M.); Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210 (T.E.O.); United Agriseeds, Inc., P. 0. Box 4011, Champaign, Illinois 61820 (G.M.P.); Department of Natural Sciences, Elmira College, Elmira, New York 14401 (P.Y.B.) ABSTRACT

seedlings (12) are induced by nitrate. Although associations have been made linking nitrate reduction and uptake (cf. 6), a number Nitrate regulation of protein synthesis and RNA translation in maize of observations support the concept that nitrate uptake is not (Zea mays L. var B73) roots was examined, using in vivo labeling with completely dependent upon nitrate reduction in higher plants [5Sjmethionine and in vitro translation. Nitrate enhanced the synthesis (22). To date, however, no identifications have been made of of a 31 kilodalton membrane polypeptide which was localized in a fraction nitrate-inducible proteins, other than NR and NiR, which might enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. be involved in the apparent induction of the uptake system. The nitrate-enhanced synthesis was correlated with an acceleration of In the present study, we have searched for nitrate-inducible net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate polypeptides in maize roots, in an attempt to identify nitrate did not consistently enhance protein synthesis in other membrane frac- membrane transport proteins. We have examined the profiles of tions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 polypeptides synthesized in maize roots incubated with or withkilodaltons) was also enhanced by nitrate. The most consistent enhance- out nitrate. The spectra of in vitro translation products of nitratement was that of the 40 kilodalton polypeptide. No consistent nitrate- induced and noninduced total root RNA were also compared. induced changes were noted in the organeliar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of 35Simethionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.

MATERIALS AND METHODS Plant Material. Maize seeds (Zea mays L. var B73) were germinated in darkness at 29'C between germination paper saturated with 0.5 mM CaSO4. After 3 d, the roots of the seedlings were transferred to aerated solutions containing 0.5 mM CaSO4, 5 mM K2HPO4, 1 mM MgSO4, 1.25 mM K2SO4, micronutrients (13) and FeEDTA at 1 mM Fe. The seedlings were grown in an incubator at 29°C under fluorescent lights (about 100-200 ,uEm-2 -s) with a light/dark cycle of 16/8 h. During induction periods, typically during the first part of the light period on the 7th d after the beginning of germination, the roots ofthe seedlings were transferred to aerated solutions containing, in addition to 10 mm Mes (pH 5.5), either 5 mM Ca(N03)2 (induced) or 5 mM CaCl2 (noninduced). To label root proteins, the roots of 5 to 8 seedlings were bathed in either control or induction solutions (45 ml) supplemented with [35S]methionine (0.2-1.0 mCi, 1100 Ci/ mmol, New England Nuclear). Nitrate uptake by replicate plants was assayed by depletion of external nitrate as described previously (20). To measure in vitro NR activity in roots of replicate plants, the extraction and assay procedures of Remmler and Campbell (24) were followed. Root Fractionation and Membrane Isolation. Roots (3-4 g) were cut into 2 to 4 cm segments and homogenized with a mortar and pestle in 35 ml cold (2-4°C) buffer plus PVPP (0.1 mg/g tissue). The homogenization buffer contained 25 mm Tris-Mes (pH 7.5), 3 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 250 mM sucrose, 100 mg/L BHT (from a stock of 50 mg/ml in isopropyl alcohol), and 5 mg/L chymostatin. The brei was filtered through Miracloth and centrifuged at 14,000g for 15 min. The pellet was resuspended in 1.5 to 2 ml of homogenization buffer minus sucrose (resuspension buffer) and stored at -70°C. The supernatant was centrifuged at 96,000g for 30 min in a Beckman SW27 rotor to obtain a microsomal pellet. Cold acetone (75 ml, 2-4°C) was added to the 96,000g supernatant (35 ml) to precip-

When roots of whole plants or plant cells, grown previously in the absence of nitrate, are first exposed to nitrate, the rate of nitrate uptake accelerates for a period of approximately 8 to 10 h. This observation has been noted in a number of higher plant species (5, 13, 15). The development of the accelerated nitrate uptake rate is limited by general inhibitors of RNA and protein synthesis (1 5) and by p-fluorophenylalanine, an analog of phenylalanine which produces ineffective protein (21). In general, it is thought that the nitrate transport system in plants is nitrateinducible, requires continual protein synthesis for maximal function, and is subject to fairly short-term turnover (9). The de novo syntheses of NR2 in barley (26), Neurospora crassa (1), and cultured tobacco cells (31), and of NiR in pea ' Conducted under the auspices of the Crop Science Laboratory, Allied-Signal Corp., P. 0. Box 6, Solvay, NY 13209. 2Abbreviations: NR, nitrate reductase; BHT, butylated hydroxytoluene; DEPC, diethylpyrocarbonate; NiR, nitrite reductase; PMSF, phenylmethylsulfonyl fluoride; PVPP, polyvinylpolypyrrolidone.

52

IDENTIFICATION OF NITRATE-INDUCIBLE PROTEINS IN MAIZE ROOTS itate the nonparticulate proteins. The precipitated proteins were collected by centrifugation and resuspended in 1.5 to 2 ml of resuspension buffer, and stored at -70°C. The membrane vesicles of the microsomal pellet were separated into three fractions by centrifugation in a discontinuous sucrose density gradient essentially as described by Leonard and Van Der Woude (16) and Booz and Travis (3). The microsomal pellet was resuspended in 5 ml of resuspension buffer containing 20% (w/v) sucrose. This was layered over 5 ml of buffer containing 34% (w/v) sucrose which was layered over 2 ml of buffer with 45% (w/v) sucrose. The gradient was centrifuged at I00,000g for 1.5 h in a Beckman SW40 rotor. Membrane vesicles sedimenting to the two interfaces (20/34%, or '20/34' and '34/45%', or '34/45') and to the bottom of the tube (45% pellet, or '45') were collected with a Pasteur pipette. Based upon its density, the 20/34 fraction was likely enriched in vesicles from the tonoplast and ER (cf 25). The 34/45 fraction was taken to be enriched in plasma membrane vesicles as suggested by the work of Leonard and Van Der Woude (16). Each fraction was diluted to 12 ml with resuspension buffer plus 200 mM KCI, and centrifuged at l00,000g for 30 min. The final pellets were resuspended with buffer to an approximate protein concentration of 1 mg/ml. Protein in the membrane suspensions was determined according to Bradford (4). The resuspended membrane fractions were stored at -70°C. Isolation of Total Root RNA. All glassware used during RNA isolation was first baked at 250°C overnight, incubated for 3 h in 0.1 % (w/v) DEPC, and autoclaved for 15 min. Plastic tubes and Waring Blendor accessories were treated for 3 h in 0.1% DEPC and autoclaved. Solutions were made with DEPC-treated (0.1% DEPC for 3 h) and autoclaved water. Roots were harvested, blotted, and immediately frozen and ground in liquid N2 with a stainless steel Waring Blendor. Five g of the powdered roots were thawed in 10 ml of 4 M guanidine isothiocyanate, 10 mm sodium citrate (pH 7.0), and 50 mM DTT, and ground again with a mortar and pestle. The brei was centrifuged for 10 min at 12,000g. Solid CsCl was added to the supernatant (1 g/2.5 ml) before layering on a 3.4 ml pad of 5.7 M CsCl, 100 mm sodium EDTA (pH 7.5). After centrifugation in a SW40 rotor for 19 h at 19°C and 225,000g, the pellet was resuspended in buffer (50 ,ul per g root tissue) containing 100 mm sodium acetate (pH 5.5), 10 mm sodium EDTA, 1% (w/v) SDS, and 50 units RNasin/ml (Boehringer Mannheim). The suspension was extracted twice with an equal volume of phenol:chloroform (1: 1, v/v) and twice with chloroform, according to Maniatis et al. (18). The RNA was precipitated (overnight at -20°C or for 1 h at -70°C) by addition of 2.5 volumes of 95% ethanol, pelletted by centrifugation in an Eppendorf centrifuge, suspended in 4 M sodium acetate (pH 5.5), incubated 10 min at 4°C, and centrifuged. The pellet was resuspended in 0.2 M sodium acetate, precipitated again with 95% ethanol, centrifuged, and washed with 0.2 M potassium acetate. After another 95% ethanol precipitation, the RNA pellet was washed two times in 70% ethanol. The final Id per g root tissue), pellet was resuspended in redistilled H20 (10 and stored in small aliquots at -70°C. A at 260 nm provided the estimation of RNA content: Mg RNA = o.d. 260 nm x 40. The 260/280 ratio for maize total RNA preparations was in the range of 1.6 to 1.8. This isolation procedure recovered 60% of standard globin RNA (purchased from Bethesda Research Laboratories). In Vitro Translation of Total Root RNA. Total root RNA was translated in a rabbit reticulocyte lysate system purchased from Bethesda Research Laboratories. Five ,ug of total RNA were added to 30 Ml of rabbit reticulocyte lysate containing 25 mM Hepes (pH 7.2), 48 mm KCI, 87 mm potassium acetate, 1.2 mm MgC12, 17 AM EDTA, 23 mm NaCl, 0.17 mM DTT, 8 AM hemin, 17 tsg/ml creatine kinase, 10 mM creatine phosphate, 50 Mm each of 19 amino acids, 600 nM (2 uCi) [35S]methionine, 0.3 mM CaC12, and 0.65 mm EGTA. The reaction mix was incubated for

53

1 h at 30°C, and then frozen at -70°C. Aliquots (4 Ml) of the reaction mix were spotted on Whatman 3MM filter discs. The discs were then washed once in 10% (v/v) TCA at 90°C for 10 min, once in cold (2-4°C) 10% TCA for 10 min, three times in 5% TCA for 2 min each, and two times in 95% ethanol. Radioactivity in the dried filters was determined by scintillation spectrophotometry. Processing of the Translation Products. After translation, the rabbit reticulocyte lysate reaction mix was centrifuged at 4°C for 1 h at 140,000g, to pellet the ribosomes. The supernatant was chromatographed on a column (0.7 x 15 cm) of Sephadex G25 to 50 which was eluted with 150 mM KCI, 20 mM Hepes (pH 7.4). The translation products were collected in the void volume, and incubated (3 x 106 cpm per reaction) for 30 min at 28°C with nonlabeled 20/34 root membranes (100 ,g membrane protein per reaction) in 0.6 M mannitol, 20 mM Hepes (pH 7.0), and 1 mM DTT. To examine if an ATP regenerating system influenced any processing, 1 mm ATP, 5 mM MgCl2, 5 mM Penolpyruvate, and 10 units of pyruvate kinase (Sigma) were added to the buffer. The reaction mixture was stored frozen at -70°C until electrophoretic analysis was performed. SDS-PAGE. Membrane samples were diluted (1:1, v/v) with resuspension buffer containing 10% (w/v) SDS and 25% (v/v) glycerol, boiled for 3 min, and centrifuged at about 10,000g for 10 min before loading onto gels. Other samples from in vitro translation experiments were boiled for 3 min in one volume of 0.06% (w/v) bromophenol blue, 15% (v/v) ,8-mercaptoethanol, 4.5% (w/v) SDS, and 240 mM Tris-HCl (pH 6.9), and one volume of 25% (w/v) glycerol, 40 m M Tris-HCl (pH 6.9). Samples (5 x 105 cpm, equivalent to 50-75 Ag protein) were loaded on 5% stacking gels and electrophoresed through 8 to 15% or 10 to 20% acrylamide gradient slabs according to the procedure of Weber and Osborne (30). Gel lanes to be compared were loaded with equal amounts of radioactivity. Protein markers (Bio-Rad mol wt markers) were electrophoresed in parallel. The gels were then stained with Coomassie blue, impregnated with fluor (Enhance, New England Nuclear), dried under vacuum with heat, and fluorographed on Kodak XAR-5 film.

RESULTS After 3, 6, or 10 h exposures to 10 mm nitrate and [35S] methionine, the relative amount of radioactivity in a 31 kD polypeptide in the 20/34 membrane fraction of roots was greater than in the corresponding region of SDS-PAGE profiles from the same membrane fraction of roots which had been exposed to [35S]methionine (for 6 h), but never to nitrate (Fig. lA). Exposure of roots to 10 mm nitrate for periods of up to 10 h caused enhanced labeling of four polypeptides (21, 40, 90, and 168 kD) found in the soluble fraction of roots (Fig. 1 B). The most consistent (i.e. across labeling experiments) enhancement in the soluble fraction was for the polypeptide at 40 kD. Comparison of nitrate-induced and noninduced SDS-PAGE profiles from the other two membrane fractions (34/45 and 45) revealed no consistent differences in labeling (data not shown). Also, no consistent nitrate-induced changes were noted in the polypeptide profiles from organellar fractions (14,000g pellet of root homogenates; data not shown). Net nitrate uptake rates of noninduced, 7-d-old replicate plants were measured at intervals through a 6.25 h period of exposure to 1.0 mm nitrate. The rates increased during this period from 3 ,Mmol nitrate -g' fresh weight h-' during the first interval of 1.75 h to 10 Mmol nitrate * g-' fresh weight * h-' during the final interval measured (Table I). Measurement of NR activities in replicate plants showed that a 6 h exposure to 10 mM nitrate significantly increased in vitro root NR activity. No NR activity could be measured in extracts of control, noninduced roots, while a NR activity of up to 11

54

Plant Physiol. Vol. 84, 1987

MCCLURE ET AL.

A

CIO v a:

'0

0 (0 '0

B

c

._

2 0 092

CO ._

-

la

(0 ID

0 VI'

20 0-

9 2-

66-6M

66

4 5-

4 5-

-

3 13 1-

2 1 1 4-

2 1.

.

40ow,

-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .t:

SE

ft,,.

FIG. 1. Fluorograms of in vivo labeled polypeptides from 20/34 membranes (A) and soluble fractions (B) of roots incubated with [35S]methionine for 6 h in the absence of nitrate (nid) or in the-presence of 10 mM nitrate for 3, 6, or 10 h (id3, id6, or id'0, respectively). Position and mol wt (x 10-3) of markers are noted on the left in each panel.

Table I. Net Nitrate Uptake Rates of Noninduced, Seven-day-old Maize Seedlings at Intervalsfollowing First Exposure to 1 mm Nitrate The roots of 8 intact seedlings (between 2.1-2.6 g fresh weight roots) were bathed in 145 ml of aerated solution initially containing 0.5 mm Ca(NO3)2 and 5 M Mes (pH 5.5). Rates were determined by measuring depletion of nitrate in the bathing solution. Values represent the means of three replicate units of eight seedlings. Standard deviations were smaller than 1 umol NO3- *g ' fresh weight. h-' for each mean. Interval Net Nitrate Uptake Rate umol NO3-g-' fresh wt-h' h Not detected *0-0.75 3 0-1.75 6 1.75-3.25 8 3.25-5.0 5.0-6.25 10

nmol N03- reduced- min' mg- protein was measured in induced root extracts. Longer exposure of roots to higher nitrate concentrations increased NR activities even further: 49 and 63 nmol N03 reduced.min-'.mg-' protein for extracts of roots exposed to 35 mm for 8 and 18 h, respectively. To examine the turnover of the nitrate-inducible, 31 kD membrane protein, roots of intact seedlings were exposed to [35S] methionine for 6 h in the presence or absence of 10 mm nitrate. The roots of some of the induced seedlings were then transferred

to aerated solutions of 5 mm CaC12, and incubated in the absence of both external nitrate and [35S]methionine for periods of up to 26 h. Fluorography of the in vivo labeled polypeptide profiles from the 20/34 membrane fractions showed that enhanced accumulation of [35S]methionine in the 31 kD polypeptide persisted in the absence of external nitrate for 26 h (Fig. 2). Total RNA was isolated from roots of 7-d-old maize seedlings exposed to 10 mm nitrate for 6 h. Chloride replaced nitrate in the noninduced controls. Translatable RNA was calculated to comprise about 0.12% of the total RNA isolated from maize roots, based upon the translation capability of purified globin mRNA. A complex array of in vitro translation products were observed by fluorography of one-dimensional SDS-PAGE profiles (Figs. 3 and 4). Apparent mol wt ranged from approximately 14 kD to above 100 kD, thus suggesting that the translatable RNA suffered little damage during isolation. The labeling of six in vitro translation products (with apparent mol wt of 21, 24, 41, 56, 66, and 90 kD) was enhanced by exposure of the roots to nitrate. The most consistent, nitrate-induced differences were at 21, 41, and 90 kD. Nitrate treatment, however, did not enhance accumulation of label in any translation products in the vicinity of 31 kD. Processing of labeled translation products with nonlabeled 20/34 membrane fractions caused changes in the SDSPAGE profiles in the 31 kD region (Fig. 4). There appeared to be a loss or a shift of a translation product of mol wt slightly smaller than 31,000, and the appearance of a new polypeptide

IDENTIFICATION OF NITRATE-INDUCIBLE PROTEINS IN MAIZE ROOTS

NID

20/34 id

nid

d

6

d Id

55

ID

26

200I

--III ti.

V

-I'm

.:f

92-

Aft...

66-

45-

45-

31-

Muftldawl .1.

`4,4--"NNW Inw,

low

312 1-

14-

ImmkidL "PAPNIMW -.

21-

r

3 Xs.

FIG. 2. Fluorogram of in vivo labeled polypeptides from 20/34 membranes of roots incubated with [35S]methionine for 6 h in the presence (id) or absence (nid) of 10 mm nitrate, followed by incubation without external [35S]methionine or nitrate for 6 or 26 h (did6 or did26, respectively). Position and mol wt (x 10-3) of markers are noted on the left.

-,l ;-.i-"'l,!:e. FIG. 3. Fluorogram of in vitro labeled translation products of total RNA isolated from roots never exposed to nitrate (NID) and those exposed to 10 mm nitrate for 6 h (ID). Position and mol wt (x10-') of markers are noted on the left.

fractions (data not shown), which should be enriched in vesicles derived from plasma membranes (16, 25). Although the inability to observe nitrate-inducible polypeptides in a plasma membraneof mol wt close to, but slightly greater than, 31,000. Addition of enriched fraction cannot be used to refute the above hypothesis, an ATP regenerating system did not affect the results (data not our results suggest that a less dense membrane fraction (of shown). putative tonoplastic enrichment and possibly containing vesicles from the golgi apparatus and/or ER (25]) contains a nitrateinducible protein involved in the regulation of nitrate uptake. DISCUSSION In barley roots, '3NO3 measurements have shown that plasma This report represents the first published identification of a membrane influx is insensitive to various nitrate pretreatments nitrate-inducible, membrane-associated protein (mol wt = designed to affect net nitrate uptake by affecting bulk tissue 31,000, Figs. IA and 2) in roots of higher plants. Because this nitrate levels (11). The results suggest that negative feedback polypeptide is associated with a membrane fraction, it is unlikely regulation of nitrate uptake originates from the tonoplast (or to be related to either of the readily soluble enzymes NR (mol some other membrane of a compartment that is interior to the wt = 116,000; 7) or NiR (mol wt = 68,000; 12), or any of their plasma membrane) rather than the plasma membrane, and probreakdown products. The correlation between the acceleration vide indirect support for the concept of a tonoplast or ER of net nitrate uptake ( 14; Table I) and the enhanced net synthesis component playing a role in the regulation nitrate uptake during of the 31 kD protein (Fig. IA) during exposure of nitrate suggests initial exposure to nitrate. Other experiments with barley (10) that this membrane protein may perform a role in the regulation and maize (26), however, have shown that short periods of nitrogen starvation increase the rate of net nitrate uptake preof nitrate uptake. Evidence that the nitrate-inducible portion of nitrate uptake dominately via increases in plasma membrane influx rate when by higher plants is dependent upon protein synthesis (15, 21) has the nitrate supply is restored. Thus, the regulation of influx of led Jackson and Volk and their associates to hypothesize that nitrate in plant roots can apparently originate either directly at the increased synthesis of a transport protein(s) in the plasma the plasma membrane or at membranes of compartments intemembrane of root cells may cause the observed acceleration of rior to the plasma membrane, depending upon the particular nitrate uptake upon initial exposure to nitrate. This hypothesis, conditions. Turnover of the 31 kD nitrate-induced, membrane protein although attractive in its simplicity, is not completely supported by the results presented here. Using either [35S]methionine label- appeared very slow during periods of external nitrate starvation ing or conventional staining (Coomassie blue or silver) methods, (up to 26 h) after a 6 h induction with 10 mm nitrate (Fig. 2). Nitrate uptake activities were not measured in replicate plants, we have not observed consistent nitrate-induced changes in SDSPAGE profiles of polypeptides from maize root 34/45 membrane however. Previous studies of nitrate uptake in maize seedlings

56

MCCLURE ET AL.

Nitrate enhanced the synthesis of four soluble polypeptides (Fig. lB). It also increased the in vitro activity of root NR (cf "Results" section). However, even though nitrate induces the synthesis of both NR (24, 26) and NiR (1 1), their mol wt or that of their subunits did not match any of the areas of enhanced labeling on our protein profiles. The most consistent nitrateinduced difference was noted at 40 kD (Fig. IB), and could represent one of the two breakdown products of NR reported at 70 and 40 kD (7, 24). The application of immunochemical techniques with anti-NR and anti-NiR antibodies should facilitate the identification of the nitrate-inducible soluble polypeptides. Comparison of translation products obtained from RNA isolated from induced and noninduced maize roots showed that six translation products were labeled more extensively in induced preparations (Fig. 3). However, no nitrate-enhanced accumulation of label was observed in products with mol wt close to 31,000. A number of conceivable explanations exist for the inability to observe nitrate-enhanced label accumulation in a 31 kD in vitro translation product corresponding to the nitrateinducible, membrane protein. Three are described as follows. Nitrate may not enhance transcription of message for the 31 kD protein, but rather may enhance or activate in vivo translation (as opposed to in vitro translation) of preexisting message. Alternatively, nitrate may enhance transcription of message for the 31 kD protein, but the message may be translated as a precursor of differing mol wt. Another possibility is that the message for the 31 kD protein may not be recovered in the employed RNA isolation procedure. The data in Figure 4 favor the second explanation, but are inconclusive until the new, 'processed,' polypeptide can be confirmed as being identical to the 31 kD membrane protein. Certainly additional work is necessary to discern which, if any, of these explanations apply.

l-

,V:1

pE-IN

2009 2-

2114-

FIG. 4.

Fluorogram of in vitro translation products of total RNA from

nitrate-induced

(id) and noninduced (nid)

roots.

Lanes

designated

as

in vitro translation products of nid and Processing involved incubating labeled translation products with 20/34 membranes (see "Materials and Methods"). Position

pnid and pid represent processed id roots, respectively.

and mol wt (x

IO-')

of markers

are

noted

on

1.

2.

the left.

3. period with 0.5 mm nitrate 36C103- and '`N03_ uptake activities declined during a 6 h nitrate starvation period, thus suggesting rapid turnover of the nitrate uptake system. It is likely, however, that the seedlings in these previous studies were not as fully nitrate-loaded as the seedlings of the studies reported herein, because of the 20-fold difference in external-inducing levels of nitrate. Recently, Teyker et aL. (28) have shown that nitrate uptake activities of fully nitrate-loaded maize seedlings (7.5 mm nitrate for 6 d) continually increased after up to 36 h of external nitrate starvation. Following 36 h of starvation, the increased rates began to decline,

(20) showed that, after

a

4

or

Plant Physiol. Vol. 84, 1987

6 h induction

but remained above the initial rates for up to 72 h of starvation.

possible, in consideration of these results, that the seedling used in the present study would have displayed a net degradation of the 31 kD membrane polypeptide only after periods of nitrate deprivation longer than 26 h, possibly longer It is

roots

than 72 h. Two types of proton gradient establishing enzymes, an ATPase (27) and a pyrophosphatase (8, 29) have been characterized from tonoplast, membranes of higher plants. These enzymes are thought to drive the transport of anions, including nitrate, into the vacuole (2, 19). It is possible that the nitrate-inducible 31 kD protein represents a subunit of either of these enzymes, or one of the channel or carrier proteins proposed by Lew and Spanswick (1 7) or Blumwald and Poole (2). The mol wt reported here for the nitrate-inducible membrane protein, however, makes it an unlikely subunit of the tonoplast-type ATPase (23).

4.

5.

6. 7.

LITERATURE CITED BAHNS M, RH GARRETT 1980 Demonstration of de novo synthesis of Neurospora crassa nitrate reductase during induction. J Biol Chem 255: 690-693 BLUMWALD E, RJ POOLE 1985 Nitrate storage and retrieval in Beta vulgaris: effects of nitrate and chloride on proton gradients in tonoplast vesicles. Proc Nati Acad Sci USA 82: 3683-3687 Booz ML, RL TRAVIS 1980 Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots. Plant Physiol 66: 1037-1043 BRADFORD MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72: 248-254 BRETELER H, CH HANISCH TEN CATE, P NISSEN 1979 Time course of nitrate uptake and nitrate reductase activity in nitrogen depleted dwarf bean. Physiol Plant 47: 49-55 BuTz RG, WA JACKSON 1977 A mechanism for nitrate transport and reduction. Phytochemistry 16: 409-417 CAMPBELL WH 1986 The biochemistry of higher plant nitrate reductase. In PW Ludden, JE Burris, eds, Nitrogen Fixation and CO2 Metabolism. Else-

vier, Amsterdam, pp 143-151 8. CHANSON A, J FICHMANN, D SPEAR, L TAIZ 1985 Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles. Plant Physiol 79: 159-164 9. CLARKSON DT 1986 Regulation of the absorption and release of nitrate by plant cells: a review of current ideas and methodology. In H Lambers, J Neeteson, I Stulen, eds, Fundamental, Ecological and Agricultural Aspects of Nitrogen Metabolism in Higher Plants. M Nijhoff and W Junk, Den Haag, Netherlands, pp 3-28 10. DREW MC, RB LEE, DT CLARKSON 1986 Effect of plant N-status on kinetics of nitrate influx measured with nitrogen- 13. Plant Physiol 80: S-89 11. GLASS ADM, RG THOMPSON, L BORDELEAU 1985 Regulation of NO3- influx in barley. Studies using '3N03-. Plant Physiol 77: 379-381 12. GuPTA SC, L BEEVERS 1985 Regulation of nitrate reduction. In JE Harper, LE Schrader, RW Howell, eds, Exploitation of Physiological and Genetic Variability to Enhance Crop Productivity. American Society of Plant Physiologists, Rockville, MD, pp 1-1 1 13. HEIMER YM, P FILNER 1971 Regulation of the nitrate assimilation pathway in cultured tobacco cells. Biochim Biophys Acta 230: 362-372 14. HOAGLAND DR, DI ARNON 1950 The water culture method for growing plants without soil. Calif Agric Exp Stn Circ 347 15. JACKSON WA, D FLESHER, RH HAGEMAN 1973 Nitrate uptake by dark-grown

IDENTIFICATION OF NITRATE-INDUCIBLE PROTEINS IN MAIZE ROOTS seedlings: some characteristics of apparent induction. Plant Physiol 51: 120-127 LEONARD RT, WJ VAN DER WOUDE 1976 Isolation of plasma membranes from corn roots by sucrose density centrifugation. Plant Physiol 57: 105114 LEW RR, RM SPANSWICK 1985 Characterization of anion effects on the nitratesensitive ATP-dependent proton pumping activity of soybean (Glycine max L.) seedling root microsomes. Plant Physiol 77: 352-357 MANIATIs T, EF FRITSCH, J SAMBROOK 1982 Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY MARTINOIA E, MJ SCHRAMM, G KAISER, U HEBER 1986 Transport of anions in isolated barley vacuoles. I Permeability to anions and evidence for a C1uptake system. Plant Physiol 80: 895-901 MCCLURE PR, TE OMHOLT, GM PACE 1986 Anion uptake in maize roots: interactions between chlorate and nitrate. Physiol Plant 68: 107-112 MORGAN MA, RJ VOLK, WA JACKSON 1985 p-Fluorophenylalanine-induced restriction of ion uptake and assimilation by maize roots. Plant Physiol 77: 718-721 MORGAN MA, WA JACKSON, RJ VOLK 1985 Uptake and assimilation of nitrate by corn roots during and after induction of the nitrate uptake system. J Exp Bot 36: 859-869 corn

16. 17.

18. 19. 20.

21. 22.

57

23. RANDALL SK, H SZE 1986 Properties of the partially purified tonoplast H+pumping ATPase from oat roots. J Biol Chem 261: 1364-1371 24. REMMLER JL, WH CAMPBELL 1986 Regulation of corn leaf nitrate reductase. II Synthesis and turnover of the enzyme's activity and protein. Plant Physiol 80: 442-447 25. ROBINSON DG 1985 Plant Membranes: Endo- and Plasma Membranes of Plant Cells. John Wiley & Sons, New York 26. SOMERS DA, TM Kuo, A KEINHOFs, RL WARNER, A OAKS 1983 Synthesis and degradation of barley nitrate reductase. Plant Physiol 72: 949-952 27. SzE H 1985 H+-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 36: 175-208 28. TEYKER RH, WA JACKSON, RJ VOLK, RH MOLL 1986 Changes in nitrate influx and efflux in two preloaded maize inbreds after transfer to nitrate-free media. Plant Physiol 80: S-28 29. WAGNER GJ, P MULREADY 1983 Characterization and solubilization of nucleotide-specific Mg+2-ATPase and Mg2'-pyrophosphatase of tonoplast. Biochim Biophys Acta 728: 267-280 30. WEBER K, M OSBORNE 1969 The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244: 4406-4412 31. ZIELKE HR, P FILNER 1971 Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J Biol Chem 246: 1772-1779