identification receptor protein in cotton fibers herbicide · identification ofareceptorproteinin...

Plant Physiol. (1987) 84, 415-4200032-0889/87/84/041 5/06/$0 1.00/0

Identification of a Receptor Protein in Cotton Fibers for theHerbicide 2,6-Dichlorobenzonitrile

Received for publication December 31, 1986 and in revised form February 27, 1987

DEBORAH P. DELMER*', STEPHEN M. READ, AND GEOFFREY COOPER2ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, California 94568-2685

ABSTRACT

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and ap-parently specific inhibitor of cellulose synthesis in higher plants. Wehave synthesized a photoreactive analog ofDCB (2,6-dichlorophenylazide[DCPAI) for use as an affinity-labeling probe to identify the DCB receptorin plants. This analog retains herbicide activity and inhibits cellulosesynthesis in cotton fibers and tobacco cells in a manner similar to DCB.When cotton fiber extracts are incubated with IHIDCPA and exposed toultraviolet light, an 18 kilodalton polypeptide is specifically labeled.About 90% of this polypeptide is found in the 100,000g supernatant, theremainder being membrane-associated. Gel filtration and nondenaturingpolyacrylamide gel electrophoresis of this polypeptide indicate that it isan acidic protein which has a similar size in its native or denatured state.The amount of 18 kilodalton polypeptide detectable by IHiDCPA-labeling increases substantially at the onset of secondary wall cellulosesynthesis in the fibers. A similar polypeptide, but of lower molecularweight (12,000), has been detected upon labeling of extracts from tomatoor from the cellulosic alga Chara corallina. The specificity of labeling ofthe 18 kilodalton cotton fiber polypeptide, coupled with its pattern ofdevelopmental regulation, implicate a role for this protein in cellulosebiosynthesis. Being, at most, only loosely associated with membranes, itis unlikely to be the catalytic polypeptide of the cellulose synthase, andwe suggest instead that the DCB receptor may function as a regulatoryprotein for f-glucan synthesis in plants.

Recent advances in genetic engineering of plants have estab-lished that directed modification of gene expression in higherplants is possible. Increasing efforts are being directed at identi-fying suitable target genes, the modification of which might leadto creation of plants with improved agronomic properties orwhich might exhibit novel forms of gene expression useful forgaining insights into basic metabolic process. The cell wall ofplants, and particularly the polymer cellulose within that wall,represent a major sink for reduced carbon. Lacking useful mu-tants blocked in various aspects of cell wall synthesis, we knowlittle about the flexibility of higher plants for surviving variousmodifications in wall structure. As an example, it would beuseful to know to what extent cellulose:lignin ratios might bemodified to enhance the quality of wood or to improve digesti-bility in forage crops. Changes in wall structure might also beexpected to alter susceptibility of plants to disease or to modifygrowth characteristics. At present, only one gene responsible for

'Present address: Department of Botany, Institute of Life Sciences,The Hebrew University, Jerusalem, Israel.

2Present address: Plant Cell Biology Research Centre, School ofBotany, University of Melbourne, Parkville, Victoria 3052, Australia.

synthesis of a cell wall component, that for the hydroxyproline-rich protein extensin, has been identified and cloned (4, 5). Nogenes involved in synthesis of the carbohydrate polymers of thewall have been identified, due primarily to the limited informa-tion available concerning the enzymes involved in plant cell wallsynthesis (10). In the case of the most abundant polymer, cellu-lose, it has not yet been possible even to demonstrate convincingsynthesis of this glucan in vitro (9). This report describes astrategy, alternative to enzyme characterization, for the identifi-cation of a polypeptide which may be involved in cellulosesynthesis in higher plants.The herbicide DCB3 is now recognized as an effective and

apparently specific inhibitor of cellulose synthesis in algae andhigher plants (10). Applied in vivo in micromolar concentrations,DCB inhibits cellulose synthesis with little or no short-termeffects on synthesis of noncellulosic polysaccharides (2, 17, 26,28), nuclear division orDNA synthesis (15, 25), protein synthesis(15), respiration (26), or the in vivo labeling patterns of UDP-glucose, phospholipids, and nucleoside mono-, di-, and triphos-phates (DP Delmer, unpublished data). It does inhibit cell wallregeneration and cytokinesis in protoplasts, presumably becausethese events require cellulose synthesis (15, 25), although a recentreport indicating some effects on cytoplasmic organization inroot hairs might indicate an effect on the cytoskeleton as well(23). DCB does not inhibit the synthesis in vitro by plant mem-brane preparations of large amounts of (1-.3)-(l-glucan or smallamounts of (1--*4)-f3-glucan (2, 8), but these activities may notrepresent reactions involved in cellulose synthesis in vivo (9).

Since DCB appears to be so effective and specific in its modeof action, we reasoned that it might interact specifically withsome polypeptide critically involved in cellulose synthesis. Wetherefore synthesized a photoreactive analog ofDCB, DCPA, foruse as an affinity-labeling reagent to detect such a receptor; underUV illumination, DCPA will decompose to a reactive nitrene,which should react and form a covalent link with any adjacentmolecule. Preliminary experiments (8) indicated that [3H]DCPAreacts specifically with an 18 kD polypeptide upon UV illumi-nation of crude cotton fiber extracts. No labeling was observedif UV illumination was omitted, and labeling was substantiallyreduced in the presence of unlabeled DCB. We report here amore detailed characterization ofthis apparent receptor for DCBin plants and discuss possible roles for this polypeptide in theprocess of cellulose synthesis in vivo.

MATERIALS AND METHODSMaterials. Cotton (Gossypium hirsutum Acala SJ-2) was

grown in growth chambers as described by Meinert and Delmer(24). Cotton ovules with their associated fibers were cultured asdescribed by Beasely and Ting (1). Suspension cultures of Nico-

'Abbreviations: DCB, 2,6-dichlorobenzonitrile; DCPA, 2,6-dichloro-phenylazide.

415

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Plant Physiol. Vol. 84, 1987

tiana tabacum L., line XD, were obtained from P. Filner of thisInstitute and grown on M-I-D medium (12). Suspension culturesof Lycopersicon esculentum VF-36 were obtained from B. Wil-liams ofthis Institute and grown on a high-salt medium describedby DuPont et al. (1 1). Both cell cultures were grown in the darkat 28°C on reciprocal shakers. Chara corralina was obtainedfrom W. J. Lucas, University of California, Davis, and grown asdescribed by Lucas and Shimmen (21). DCB was obtained fromFluka, Sepharose 4B from Pharmacia, and other standard re-agent-grade chemicals were from Sigma. 2,6-Dichloro-[3H]ani-line was prepared on special request by New England NuclearCorporation. U-['4C]glucose, [14C]-labeled protein mol wt stan-dards, and Amplify were obtained from Amersham.

Preparation of IHJDCPA. 2,6-Dichloro-[3H]aniline was puri-fied by HPLC, converted to its diazo salt, and then reacted withsodium azide to produce [3H]DCPA as described in detail byCooper et al. (6). Following purification by HPLC, the [3H]DCPA, in methanol, had a specific activity of approximately 2.4Ci/mmol. Unlabeled DCPA was synthesized and purified simi-larly.[3H]DCPA is stable for many months when stored in thedark in methanol at 4C. The slow rate of decomposition (ap-proximately 10% after several months) was monitored by TLCin the dark in n-hexane; decomposition products remained at ornear the origin, while DCPA migrated at anRF of about 0.4. Wehave recently obtained some evidence for loss of tritium viaexchange with protons in aqueous solution at low pH (<3).

Effects of DCB and DCPA in Vivo. Cotton ovules, with theirassociated fibers, were cultured for 17 d post-anthesis. The ovulesand fibers were carefully removed from flasks and drained onpaper towels, rinsed in culture medium lacking glucose, and 4ovules per incubation were placed in 2 ml of the same mediumlacking glucose with or without DCB or DCPA. DCB and DCPAwere dissolved in dimethylsulfoxide and diluted into the mediumto give the appropriate concentration. All incubations, includingcontrols, were then adjusted to give a final concentration ofdimethylsulfoxide of 0.4% (v/v). Following preincubation in thedark for 15min at30°C,[14C]glucose (1.3mM; 0.65 Ci/mol) was

then added and incubation continued for 90min. The ovuleswere then removed and washed in medium lacking glucose, andthe fibers were removed from the ovules, frozen, and lyophilized.After weighing, noncellulosic material was solubilized by heatingthe fibers in acetic-nitric reagent and radioactivity in the insolu-ble cellulosic residue was determined as described in detail byMontezinos and Delmer (26). Tobacco cells, harvested 5 d aftersubculture, were filtered by gravity on Whatman No. 4 paper

and washed 3 times with M-I-D medium lacking glucose. Ali-quots (300 mg fresh weight) were then preincubated with or

without inhibitors, and subsequently incubated with['4CJglucose(1.9 mm; 2.8 Ci/mol) as described above. The cells were thencollected by filtration on Whatman GF/A filters, washed inwater, and radioactivity in cellulose determined as for cottonfibers.

Labeling of Extracts with HI3HDCPA. Except where indicated,all extracts of cotton fibers were prepared using locules whichwere frozen in liquid N2 immediately following harvest frombolls taken at 19 to 22 d post-anthesis. (Similar results wereobtained using fibers harvested without freezing of locules.)Frozen fibers were removed from ovules and ground to a powderin liquid nitrogen. They were then extracted in a chilled mortarand pestle with 25 mm Hepes/KOH (pH 7.3) containing 2.5 mmMgCl2, 0.5 mm phenyl methylsulfonyl fluoride, and 0.05 mmleupeptin, using 2 ml buffer for fibers derived from each loculeharvested. The extract was then filtered through three layers ofMiracloth. Labeling was either performed at this time or follow-ing centrifugation of the extract at4°C for 45min at100,000gusing a Beckman 80 Ti rotor. Membranes were resuspended to

theiro riginalvolume by homogenization in the extraction buffer

described above. Although labeling was routinely performedimmediately, recent experiments show that similar results can beobtained with extracts which have been stored at -80°C. Extractsof suspension-cultured tomato cells were prepared similarly,except that cells were harvested by gravity filtration on WhatmanNo. 4 paper, washed in extraction buffer lacking protease inhib-itors, and then disrupted in extraction buffer containing MgCl2and inhibitors, using a French Press at 6000 p.s.i. Chara extractswere also prepared similarly using washed internode cells whichwere in the final stages of elongation; they were disrupted bychopping with scissors.To label a crude extract,1 00,000g supernatant, or membranes,

under dim light, 25 ul of[3H]DCPA in methanol (2,uCi; approx-imately 0.8Mm final concentration) were added per ml of sample,and this mixture was incubated in the dark at4°C for 10 to 15min. Samples were then placed in small plastic Petri dishes at4°C and illuminated for 5 min using a Mineralight Short-WaveUV lamp (UVP Inc.) placed 3 cm above the sample, giving anintensity of approximately 400 to 500 MW/cm2. (Analysis byTLC of the time course of decomposition of[3H]DCPA underthese conditions of illumination indicates that complete decom-position occurred within 1-2 min.) Any remaining reactivespecies were then quenched by addition of DTT to mm. (Wehave recently shown that omission of this step gives similarresults.)

Analysis of Labeled Samples by PAGE. For each lane analyzedby PAGE, a labeled sample (0.5 ml labeled crude extract, super-natant, or membranes containing 100-300 Mg protein) was pre-

cipitated in four volumes of cold acetone containing 9 mm aceticacid and stored for at least 2 h at-20°C. The resulting precipitatewas collected by centrifugation at 4000g for 5 min and resus-

pended with sonication in 25 to 50 ul of 0.125 Tris-HCl (pH6.8) containing 50mM DTT and 10% (v/v) glycerol; samples forPAGE-SDS also contained 2% (w/v) SDS (Serva) and these were

placed in a boiling water bath for 30 sfollowing sonication. Priorto addition of DTT, protein in these samples was determinedusing a modification of the Lowry procedure (22). An aliquot of5 to 15 Ml of the sample was loaded per lane on 15% acrylamidegels prepared by the procedure of Laemmli (20) using a Hoeffervertical mini-slab gel apparatus and 0.75 mm spacers. Acrylam-ide concentration in the stacking gel was 4.5%. Nondenaturinggels were prepared in similar fashion but lacked SDS in gels andrunning buffer. Following electrophoresis at 10 to 15 mamp pergel, gels were fixed for 30min in 50% (v/v) methanol/10% (v/v) glacial acetic acid, then for 5 mm in 10% methanol/5% aceticacid. The gels were then incubated with Amplify for 15min,dried, and subjected to fluorography using Kodak X-OMAT filmat-80°C. Under optimal labeling conditions, the 18 kD bandwas usually faintly visible within 24 to 48 h; good exposures

usually required 2 weeks.

RESULTSDCB Analog DCPA Mimics DCB in Its Effects in Vivo in

Plants. Structure-function studies with the herbicide DCB haveindicated that analogs that retain a planar structure and halogengroups at positions 2 and 6 of the benzene ring retain herbicideactivity (13, 14). This indicated to us that DCPA should thereforebe a biologically active analog of DCB. Results shown in TableI confirm that unlabeled DCPA, like DCB, is an effective inhib-itor of cellulose synthesis in cotton fibers and suspension-culturedtobacco cells. On a concentration basis, DCPA is almost as

effective as the parent herbicide DCB, having a K, in the range

of 0.5 to 1,m for cotton fibers and about 10 mM for tobacco. Inaddition, extensive herbicide tests of DCPA using a variety ofcrop and weed plants indicate that DCPA is indistinguishablefrom DCB in its range and effectiveness as a pre-emergent

erbicide(DP Delmer, Cooper, unpublished data). Such

416 DELMER ET AL.



IDENTIFICATION OF RECEPTOR FOR DCB

Table I. Effects ofDCB and DCPA on Cellulose Synthesis in VivoAll incubations were performed in the dark at 30'C in appropriate

culture medium for cotton ovules and fibers (1) or tobacco cells (12), butlacking the normal carbon source. After 15 min preincubation withinhibitors, ['4Clglucose (1.9 mm, 2.8 Ci/mol for tobacco; 1.3 mm, 0.65Ci/mol for cotton) was added and the incubation continued for 90 min.Radioactivity in cellulose was measured as described in "Materials andMethods" and Montezinos and Delmer (26).

Radioactivity in CelluloseInhibitor Concentration TobaccoAdded Cotton fibers suspension

cultures

cpm/mg dry cpm/mgfreshMM wtfibers wt cells

None 1998 302

DCB 1 8953 42610 279 14320 209

DCPA 1 10873 73310 356 14720 .262

93 kd-

68 kd-

30kd-

14 kd -

Top of gel

18 kd

S M S M

Labelling done: Before or AfterCentrifugation

FIG. 1. Identification of 18 kD DCB receptor in cotton fibers. Figureshows a fluorogram of cotton fiber proteins separated by SDS-PAGEfollowing labeling with [3H]DCPA. S = supernatant obtained fromcentrifugation of crude extract at 4°C for 45 min at 100,000g. M =

membrane fraction (pellet) obtained from such centrifugation.

sults support the notion that DCPA should interact well with theDCB receptor in plants.

Identification of a DCB Receptor in Cotton Fibers. Resultsshown in Figure 1 document that [3H]DCPA preferentially in-teracts with, and upon UV illumination covalently couples to,an 18 kD polypeptide found in crude extracts of cotton fibersharvested at the time of onset of secondary wall cellulose synthe-sis, and that the majority of this polypeptide is found in thel00,OOOg supernatant of such extracts. The level of labeling ofthis polypeptide is pronounced, and we conclude it is specific forseveral reasons: for example, addition of DCB with [3H]DCPA

substantially reduces the label incorporated into this, but noother, band (8) (DP Delmer, unpublished data); and the proteinhas high affinity for DCPA since label saturates in this compo-nent, but in no other bands, at concentrations ofDCPA between1 and 5 AM (data not shown).Nonspecific labeling does occur, particularly in the membrane

fraction, as evidenced by the fact that, in terms of total radioac-tivity incorporated into all acetone-precipitable material, themembrane fraction contains about 3 times as much radioactivityas the supernatant. Upon prolonged exposure ofthe fluorograms,most of this label in the membrane fraction follows the patternof Coomassie blue staining of the membrane tracks in the gels.Such enhanced nonspecific labeling in the membrane fractionmay be due to the hydrophobic nature of DCPA and its prefer-ential partitioning into the membranes. This may also explainwhy labeling of the soluble 18 kD component is more efficientif labeling is performed after the membranes are removed bycentrifugation (see below). In addition to the nonspecific labelingin the membranes, about 10% of the total label found in the 18kD polypeptide can be demonstrated in the membrane fraction,provided that labeling is carried out prior to centrifugation ofthe crude extract. The loss of ability to label the minor 18 kDcomponent in the membrane following centrifugation could beexplained by the presence of some factor in the supernatantrequired for efficient interaction ofDCPA with its receptor. Somelabel of questionable specificity (see later section on develop-mental regulation and "Discussion") is also found in a mem-brane-associated component(s) which migrates through thestacking gel and focuses at the top of the separating gel. Inclusionofurea in the sample buffer has not resulted in enhanced mobilityof this membrane-associated component.MgCl2 is routinely added to the homogenization buffer and is

present during labeling, although its presence is apparently notabsolutely required for interaction with DCPA (Fig. 2A). How-ever, results of experiments shown in Figure 2 imply that somedivalent cation may play a role in this interaction since thepresence ofEGTA (plus Mg2") or EDTA during labeling resultsin substantial reduction in ability to label the soluble 18 kDpolypeptide, but has no effect on nonspecific labeling.

Interpretation of these results is complicated by several addi-tional findings: (a) the inhibitory effect of chelators is morepronounced when labeling is performed prior to separation ofmembrane and soluble components, and inhibition is only ob-served for the soluble, and not the membrane-associated, 18 kDcomponent (Fig. 2A); and (b) we have not been able to restorethe capacity to label it in the presence of such chelators byreaddition of Ca2", Mg2e, Mn2", or Zn2", nor have we succeededin removing any necessary divalent cation or other factor bypassage ofcrude extracts through G-25 Sephadex; thus, followingdesalting on such a column equilibrated in 25 mm Hepes/KOH(pH 7.3), the 18 kD polypeptide elutes in the void volume andcan be labeled successfully (not shown). We have noted, however,that purification steps such as DEAE-Sepharose or native gelelectrophoresis followed by electroelution do result in loss ofability to detect any fraction which can be labeled by DCPAeven though the prelabeled polypeptide can be recovered suc-cessfully from these purification steps. Thus, a possible role fora tightly bound divalent cation(s) and/or other factor in facilitat-ing interaction of DCPA with its receptor is implied but notclarified to date. Studies with divalent cations do show, however,that the presence of 30 to 50 AM Zn2` (Cl- or S042 salt; S042only shown) specifically inhibits labeling of the 18 kD solublepolypeptide (Fig. 2B).DCPA-Labeling of the 18 kD Polypeptide Parallels Rate of

Cellulose Synthesis. The fact that the majority of specific labelis found associated with a soluble polypeptide indicates that it ishighly unlikely that the 18 kD DCB receptor is a subunit of the

417




|,-- I OETA

-40T op ofSeparating Gal

0- 18 kid-I~~~

SM S M

v: Afteri

B.Tolp ofSeparsting G1l

11 ked

ZaSO4 (pM): 0 1 3 10 30 50

2 400

l CL0 02

300

2 200

0

100i .'

I CL

lo

U8 10 12 14 16 ls 20 22

_c

5 $0_E

4 8.

30%0

0

a.2 2

c

-a1 o0r_

Days post-anthesis

FIG. 3. Labeling of soluble 18 kD polypeptide in extracts derivedfrom cotton fibers at various stages of fiber development. The 100,OOOg

supernatants were prepared, labeled, and separated by SDS-PAGE as

described in "Materials and Methods." Following fluorography, the area

ofthe 18 kD polypeptide was cut from the dried gels and quantitated byliquid scintillation counting; cpm in this band were then standardized tocpm per 100 gg protein based on the original quantity of total proteinseparated in each lane of the gel. The rate of cellulose synthesis in vivo,standardized per mm fiber length, was determined as described byMeinert and Delmer (24).

FIG. 2. Effects of divalent cations and/or chelators on labeling ofDCB receptor. A, Fluorograms ofcotton fiber proteins separated by SDS-PAGE following labeling with [3H]DCPA in the absence of added diva-lent cations; or in the presence of 2.5 mM MgC12 and 5 mM EGTA; or in5 mm EDTA. For this experiment, MgCl2 was omitted from the standardhomogenization buffer. B, Similar fluorogram showing the effect ofvarious concentrations of ZnCl2 on labeling of l00,OOOg cotton fibersupernatant. MgCl2 was (2.5 mM) present in the homogenization buffer.The first, unmarked lane in this gel represents standard ['4C]proteinmarkers (STDS). S and M are defined in the legend to Figure 1.

cellulose synthase, which is presumed to be an integral compo-nent of the plasma membrane (9). However, studies on thedevelopmental regulation ofthe 18 kD polypeptide in developingcotton fibers do provide further support for the notion that it issomehow involved in the process ofcellulose biosynthesis. Thesestudies show that the ability to label this polypeptide increaseswith the increase in rate of cellulose synthesis in vivo in thesefibers (Fig. 3). Labeled 18 kD polypeptide can barely be detectedin elongating fibers when the rate of cellulose synthesis is low,and substantially increases at the onset ofsecondary wall cellulosesynthesis. In two of three experiments, we also observed a con-comitant increase in capacity to detect that labeled band in themembrane fraction which migrates at the top of the separatinggel (not shown).

Characterization of the DCB Receptor in Its Native State. Thelabeled, soluble DCB receptor was found to precipitate followingaddition of ammonium sulfate to 70% saturation (not shown).When resolubilized and subjected to gel filtration on Sepharose4B and various fractions pooled and analyzed by SDS-PAGE, itwas found that the DCB receptor is <20 kD in its native state aswell (Fig. 4, pool D). The quantity of label which elutes in thevoid volume (A) in such experiments was found to be variable,and the identity of this material remains uncertain; althoughprecipitated by ammonium sulfate, it is not precipitated inacetone. We suggest that it might represent nonspecifically la-beled polyphenolic material, which due to its hydrophobic naturemight interact well with DCPA, be soluble in acetone, and the

.3~~~~~~~~~~~~~~~~~~~~~~~13.-

T 40 IN 43 20

A B C 0 E

30 40UU

Fim Mnhw

FIG. 4. Chromatography of [3H]DCPA-labeled soluble fraction fromcotton fibers on Sepharose 4B. Labeled 100,000g supernatant was con-

centrated by ammonium sulfate precipitation and separated at 40C on

Sepharose 4B column (2.2 x 32 cm) equilibrated in 25 mM HEPES/KOH (pH 7.2). Fractions (1 ml) were collected, pooled as indicated (A-E), precipitated with acetone, and analyzed by SDS-PAGE followed byfluorography (inset to figure). Arrows indicate the positions of elution ofthe molecular size markers: urease dimer (480 kD), aldolase (158 kD),ovalbumin (43 kD), and trypsin inhibitor (20 kD).

content of which is variable in our extracts. When the pooledfractions containing the DCPA-binding polypeptide (pool D)were concentrated and passed through a G-50 Sephadex column,the 18 kD polypeptide eluted between the void volume (-30 kD)and a cytochrome c marker (mol wt 12,000).

In SDS-PAGE, the 18 kD polypeptide migrates in very closeproximity to calmodulin from spinach or cotton fibers, and weconsidered that this polypeptide might be calmodulin, which isplausible in view of the proposed modulation of callose and

A. StDTOS EGTA EOTA

93

68-45-

30-

14-

S M S M S M

Labelling Dons: Before uCentritugation

IF

[3H] - DCPA- polypeptide

< ~~~~~"Rawte of cellulose synthesie

/__l_____& . .

/1

I------.. . I. J

goo.w-

418 DELMER ET AL.

_

_



VANADATE-SENSITIVE H+-ATPase OF CORN ROOTS

one or more polypeptides, but we have not pursued this questionfurther.The initial rate ofintravesicular acidification in this membrane

fraction is 75 to 80% inhibited by 200 ,M vanadate. Half-maximal inhibition is observed at approximately 60 ,M vanadate,a value higher than that previously reported for the plasmamembrane ATPase activity of corn (10 ,uM vanadate [24]; Fig.2), but quite similar to that observed when the ATPase activitywas measured in experimental conditions close to those of theH+-pumping measurements (40 ,uM vanadate over 4 min ofincubation). The strong dependence of the inhibition of ATPaseactivity by low concentrations of vanadate on the length of theincubation period might indicate that the accessibility of vana-date to its binding site is somewhat limited in our experimentalconditions. This result could be explained by a delayed action ofvanadate on the ATPase, as observed for the Neurospora ATPaseby Bowman and Slayman (5).The effect of different anions on the rate of intravesicular

acidification seems to reflect essentially their different abilities tocollapse the A4V built up by the electrogenic transport of H+ (23,26). In fact, the initial rate of intravesicular acidification isstrongly affected by anions (Br- > Cl- = NO3- >> SO42-) whenmeasured in the absence of valinomycin, but much less so in itspresence: the stimulating effect of valinomycin increases follow-ing the sequence NO3- < Br- < Cl- << S042-, as would beexpected on the basis of the permeabilities of plant membranesfor these anions (1 1, 26).However, a more specific effect of these anions on the H+-

ATPase (23) cannot be ruled out on the basis of the availabledata.

Sensitivity to inhibitors, kinetics as a function of ATP concen-tration (apparent Km 0.7 mM), divalent cation requirement (Mg2+= Co2+, Mn2+ >> Zn2+ > Ca2+ = 0), dependence on the pH ofthe incubation medium (strict pH optimum at pH 6.5) of thevanadate-sensitive H+-pumping activity, correlate well with thosereported for the plasma membrane ATPase of corn roots (13,22), as well as of other plant materials (6, 8, 14, 23, 29, 30).These data, taken as a whole, provide strong evidence that the

plasma membrane ATPase of corn roots is a H+-pump.The inhibitors tested, with the exception of vanadate and

NO3-, do not show much specificity toward the plasma mem-brane H+-ATPase as compared to the tonoplast one. However,the two H+-ATPases can be distinguished on the basis of theirspecificity for divalent cations (Mn2+ is much more active thanCo2+ on the tonoplast H+-ATPase), apparent Km for ATP (muchlower for the tonoplast H+-ATPase) and sensitivity to the pH ofthe incubation medium (the tonoplast H+-ATPase shows a broadpH optimum around pH 7) (13, 14, 20, 22, 29).The availability of a membrane fraction enriched in vanadate-

sensitive H+-ATPase opens new possibilities for the in vitro studyof its regulation and of its implication in the transport of otherions and solutes, possibly via cotransport systems.

LITERATURE CITED

1. AMES BN 1966 Assay of inorganic phosphate, total phosphate, and phospha-tases. Methods Enzymol 8: 115-118

2. BENNETT AB, SD O'NEILL, M EILMANN, RM SPANSWICK 1985 H+-ATPaseactivity from storage tissue of Beta vulgaris. III. Modulation of ATPaseactivity by reaction substrates and products. Plant Physiol 78: 495-499

3. BENNETT AB, SD O'NEILL, RM SPANSWICK 1983 H+-ATPase activity from

storage tissue of Beta vulgaris. I. Identification and characterization of ananion-sensitive H+-ATPase. Plant Physiol 74: 538-544

4. BENNETT AB, RM SPANSWICK 1983 Optical measurements of ApH and A/ incorn root membrane vesicles: kinetics analysis of C1l effects on a protontranslocating ATPase. J Membr Biol 71: 95-107

5. BOWMAN BJ, CW SLAYMAN 1979 The effects of vanadate on the plasmamembrane ATPase of Neurospora crassa. J Biol Chem 254: 2928-2934

6. BRISKIN DP, RJ POOLE 1983 Characterization of a K+-stimulated adenosinetriphosphatase associated with the plasma membrane of red beet. PlantPhysiol 71: 350-355

7. CHANSON A, L TAIZ 1985 Evidence for an ATP-dependent proton pump onthe Golgi of corn coleoptiles. Plant Physiol 78: 232-240

8. Cocucci MC, E MARRtE 1984 Lysophosphatidylcholine-activated, vanadate-inhibited Mg2+-ATPase from radish microsomes. Biochim Biophys Acta 771:42-52

9. Cocucci MC, MI DE MICHELIS, MC PUGLIARELLO, F RASI-CALDOGNO 1985Reconstitution of proton pumping activity of a plasma membrane ATPasepurified from radish. Plant Sci Lett 37: 189-193

10. COLOMBO R, A BONETri, R CERANA, P LADO 1981 Effects of plasmalemmaATPase inhibitors, diethylstilbestrol and orthovanadate, on fusicoccin-in-duced H' extrusion in maize roots. Plant Sci Lett 21: 305-315

11. DE MICHELIS MI, MC PUGLIARELLO, F RASI-CALDOGNO, L DE VECCHI 1981Osmotic behaviour and permeability properties of vesicles in microsomalpreparations from pea internodes. J Exp Bot 32: 293-302

12. DUPONT FM, BENNETT AB, SPANSWICK RM 1982 Localization of a proton-translocating ATPase on sucrose gradients. Plant Physiol 70: 1115-1119

13. DUPONT FM, LL BURKE, RM SPANSWICK 1981 Characterization ofa partiallypurified adenosine-triphosphatase from a corn root plasma membrane frac-tion. Plant Physiol 67: 59-63

14. HAGER A, W BIBER 1984 Functional and regulatory properties of H+ pumpsat the tonoplast and plasma membrane of Zea mays coleoptiles. Z Natur-forsch 39c: 927-937

15. HAGER A, M HELMLE 1981 Properties ofan ATP-fueled, C I -dependent protonpump localized in membranes ofmicrosomal vesicles from maize coleoptiles.Z Naturforsch 36c: 997-1008

16. HARTMANN MA, E EHRHARDT, P BIENVENISTE 1983 Concanavalin A bindingas a tool in determining the sidedness of membrane vesicles from maizecoleoptiles. Plant Sci Lett 30: 227-238

17. IMBRIE CW, TM MURPHY 1984 Proton pumping by vesicles reconstitutedfrom two fractions of solubilized rose-cell plasma membrane ATPase.Biochim Biophys Acta 778: 229-232

18. LANZETTA PA, LJ ALVAREZ, PS REINACH, OA CANDIA 1979 An improvedassay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95-97

19. LEW RR, N BusHUNOW, RM SPANSWICK 1985 ATP-dependent proton-pump-ing activities of zucchini fruit microsomes: a study of tonoplast and plasmamembrane activities. Biochim Biophys Acta 821: 341-347

20. MANDALA S, IJ METLER, L TAIZ 1982 Localization of the proton pump ofcorn coleoptile microsomal membranes by density gradient centrifugation.Plant Physiol 70: 1743-1747

21. MARKWELL MAK, SM HAAS, LL BIEBER, NE TOLBERT 1978 A modificationof the Lowry procedure to simplify protein determination in membrane andlipoprotein samples. Anal Biochem 87: 206-2 10

22. O'NEILL SD, AB BENNErr, RM SPANSWICK 1983 Characterization of aN03-sensitive H+-ATPase from corn roots. Plant Physiol 72: 837-846

23. O'NEILL SD, RM SPANSWICK 1984 Characterization of native and reconsti-tuted plasma membrane H+-ATPase from the plasma membrane of Betavulgaris. J Membr Biol 79: 245-256

24. O'NEILL SD, RM SPANSWICK 1984 Effects of vanadate on the plasma mem-brane ATPase of red beet and corn. Plant Physiol 75: 586-591

25. RANDALL SK, AW RUESINK 1983 Orientation and integrity of plasma mem-brane vesicles obtained from carrot protoplasts. Plant Physiol 73: 385-391

26. RASI-CALDOGNO F, MC PUGLIARELLO, MI DE MICHELIS 1985 Electrogenictransport of protons driven by the plasma membrane ATPase in membranevesicles from radish. Biochemical characterization. Plant Physiol 77: 200-205

27. SERRANO R 1984 Purification of the proton-pumping ATPase from plantplasma membranes. Biochem Biophys Res Commun 121: 735-740

28. SZE H 1983 H+-pumping ATPase in membrane vesicles of tobacco callus:sensitivity to vanadate and K'. Biochim Biophys Acta 732: 586-594

29. SZE H 1985 H+-translocating ATPases: advances using membrane vesicles.Annu Rev Plant Physiol 36: 175-208

30. VARA F, R SERRANO 1982 Partial purification and properties of the proton-translocating ATPase of plant plasma membranes. J Biol Chem 257: 12826-12830

547



420 DELMER ET AL.

brane preparations derived from higher plants. Coupled

is the ease with which callose ([l- 3J-(-glucan)

sized in vitro by the plasma-membrane-localized, Ca2l-activatedUDP-glucose: (l- 3)-#3-glucan synthase (9). The

the callose synthase and the cellulose synthase may

enzyme has been raised several times in the past

currently considering a model (see also Ref. 9) in

UDP-glucose:glucosyltransferase exists in the plasma

of plants. In this model, the function of the 18

would be as a regulatory protein which modulates

of the glucosyltransferase with regard to the type

creates upon polymerization of glucose residues ,B-glucan.

In intact plant cells, the 18 kD polypeptide

association with the glucosyltransferase; in such

catalytic centers and acceptor glucan are aligned

as to favor transfer of new glucosyl residues to the 4-hydroxyl

the acceptor residue, resulting in synthesis of cellulose.

of DCB to its receptor results in dissociation

mation of the 18 kD polypeptide, rendering the

ase nonfunctional. Alternatively, in the absence

upon influx of Ca2" and/or cell damage, the 18

from the complex, and the glucosyltransferase assumes

conformation and functions as a callose synthase,

new glucosyl residues to the 3-hydroxyl of the

model may also provide an explanation for

regulation of the 18 kD polypeptide: changes

cellulose synthesis in vivo would be regulated, not

the level of glucosyltransferase, but rather by changes

the 18 kD polypeptide. This type of regulationallows

maintain a high level of potential callose synthase

allowing it to respond quickly upon damage.

Arguing perhaps against such a model is the general

very rigorous specificity of glycosyltransferases

the linkage formed during glycosyltransfer (30).

ever, two notable exceptions: (a) a report of a a-

fucosyltransferase which still retains the capacity

fucose in either (1--3)- or (1-}4)-linkage (29);

ulation of lactose synthase by the 14 kD acidic

lactalbumin (16). Both cases bear some resemblance

model. Binding of lactalbumin to a Golgl-localized

transferase alters the acceptor specificity of the

it prefers to transfer galactose to free glucose,

synthesis of lactose; in the absence of lactalbumin,

its induction by hormones during lactation, the

galactose to terminalGlcNAc residues on

through the Golgl. In this system, however, it

that the linkage formed in either case is the same-4(--4)-(i.Certainly the 18 kD polypeptide and lactalbumin

resemblance in size and charge properties and

developmental regulation. Clarification of the

cations in modulating interaction with DCB

clues to function. In this regard, it may be

lactalbumin is reported to contain a binding site Zn2+

inhibits labeling ofthe DCB receptor) and a affinity

site for Ca`+ (27).Obviously, other models for the function of poly-

peptide are possible. Since the cytoskeleton

involved in the orientation of cellulose microfibrils,

tide could represent some component of that

participates in cellulose synthesis. Ultimate understanding

function of this receptor must await its complete

and characterization. If this polypeptide is found

role in the regulation of the rate of cellulose

then the gene which codes for it may well be an

for genetic englneering.


Acknowledgments-We thank J. Bussell and C. Nitsche for technical assistanceand K. Long for preparation of this manuscript.

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