Plant Physiol. (1977) 59, 594-599

Auxin-binding Sites of Maize Coleoptiles Are Localized on

Membranes of the Endoplasmic Reticulum1Received for publication September 24, 1976 and in revised form November 22, 1976

PETER M. RAY2Department of Biological Sciences, Stanford University, Stanford, California 94305

ABSTRACT

Sites in maize (Zea mays L.) coleoptile homogenates that reversiblybind naphthalene-l-acetic acid with high affinity and may representreceptor sites for auxins are located primarily on ceilular membranes thatshow the enzymic and buoyant density characteristics of membranes ofthe rough endoplasmic reticulum. The sites remain attached to theendoplasmic reticulum (ER) membranes after the ribosomes have beenstripped off them. Binding sites for naphthylphthalamic acid, an inhibi-tor of auxin transport, are located on membranes different from thosethat carry the naphthalene-1-acetic-acid (NAA)-binding sites, and whichare probably plasma membrane. The two kinds of binding sites can belargely separated by appropriate density gradient centrifugation. Theresults raise the possibility that primary auxin action occurs at ERmembranes and could represent facilitation of the transfer of hydrogenions and nascent secretory protein into the ER lumen followed bysecretory transport of these products to the cell exterior via the Golgisystem.

Hertel et al. (9) and Ray et al. (24) have described andcharacterized membrane-bound sites, in maize coleoptile ho-mogenates, that bind naphthalene acetic acid and other auxins.Lembi et al. (13) similarly reported on membrane-bound bindingsites for the auxin transport inhibitors N-1-naphthylphthalamicacid and morphactins. The present paper shows the behavior, indensity gradient fractionation, of the membranous particles thatbear these sites. The results indicate that most of the NAA3-binding sites are located on membranes of the RER of these cellswhile the NPA-binding sites are located on a different mem-brane system, probably the plasma membrane. This same con-clusion was recently published (19), based on a more limited cellfractionation procedure, in work that was initiated while thepresently described study was in progress.

MATERIALS AND METHODS

Plant Material. Seedlings of Zea mays L., hybrid WF9 x Bear38, were raised, harvested for coleoptiles, and homogenized, asdescribed previously (24). The homogenization medium was asspecified (24) except in certain experiments noted below inwhich additional Mg2+ was included.

I This work was supported by grants to R. Hertel from the DeutscheForschungsgemeinschaft (SFB 46) and to P. M. R. from the NationalScience Foundation.

2 Much of this work was done while the author was on sabbatical leaveat the Institut fuir Biologie III, Universitfit Freiburg im Breisgau, Ger-many.

3Abbreviations: ER: endoplasmic reticulum; RER: rough endoplas-mic reticulum; NAA: naphthalene-1-acetic acid; NPA: N-1-naphthyl-phthalamic acid.

Differential Centrifugation. The homogenate was centrifugedin an angle rotor (Sorvall SS-34 up to 41,000g, Spinco type 65for higher forces) in the appropriate centrifuge operated at 0 C.Pellets were washed by resuspension in 0.25 M sucrose contain-ing 10 mm tris (pH 7), 1 mm EDTA, and 0.1 mM MgCl2. Analiquot was removed for enzyme assays and the remainder ofeach particle fraction was recentrifuged exactly as in the centrif-ugation by which it had been previously pelleted. Each pelletwas resuspended, using a Potter-Elvehjem type homogenizer,using 1.8 ml of standard binding assay medium (pH 5.5) (24) perg fresh weight of tissue from which the particles were derived.This suspension was used for binding assays.

Rate Zonal Centrifugation. The homogenate was centrifuged10 min at 5OOg to remove any debris, then 10 ml of it waslayered over each of two linear 30-ml gradients of 10 to 25% (w/w) sucrose made up in 10 mm tris (pH 7), containing 1 mMEDTA, 0.1 mM MgC92, and 1 mm KCI (gradient basal medium).The gradients, each contained in a cellulose nitrate tube (3.1 x8.9 cm), were centrifuged 30 min at 9,000 rpm in the Spinco SW25.2 rotor (10,OOOg at ray), and fractionated from the bottominto 4.2-ml fractions. Corresponding fractions from the twogradients were combined, centrifuged 20 min at 100,000g, thepellet was resuspended in 3.5 ml of 10% sucrose containing thesame constituents as the gradient media just listed, and used forthe assays to be described.

Isopycnic Centrifugation. The majority of the mitochondria,plastids, and larger particles were removed by preliminary cen-trifugation of the homogenate in an angle rotor for 10 min at6780g. Particles in the supernatant from this centrifugation wereconcentrated by layering 49 ml of it over layers of 6 ml 45% (w/w) sucrose and 6 ml 10% sucrose in gradient basal medium,contained in a tube (3.1 x 8.9 cm). This was centrifuged 35 minat 25,000 rpm in the Spinco SW 25.2 rotor. The tube waspierced at the bottom and from the effluent, collected dropwise,about 5 ml corresponding to the 10%/45% sucrose boundarywas selected, diluted until its sucrose concentration was below15% (w/w), and layered over a 52.5 ml, 15 to 45% (w/w) lineargradient made up in gradient basal medium. This was centri-fuged usually for 3 hr at 25,000 rpm in the SW 25.2 rotor(75,000g at ra,). Runs made for 16 hr showed the same distribu-tion of binding activity and marker enzymes as at 3 hr, so thegradient is known to be isopycnic at that time. The gradient wasfractionated into 3.6-ml fractions, which were used for enzymeassays directly and for binding assays as described below.

In experiments involving high Mg2+, the homogenization me-dium contained 4 mm MgCl2, and all other media in the densitygradient procedure contained 3 mM MgC92, in addition to theother constituents indicated above for these media.

Stripping of ribosomes from isolated RER membranes wasdone by pooling fractions of 31 to 37% sucrose from two highMg2+ isopycnic gradients prepared as above, and diluting withtwice their volume of 6 mm EDTA in gradient basal medium.This was layered over a second 15 to 45% sucrose (16.5 ml)gradient made up in gradient basal medium, centrifuged for 1.5

594

LOCALIZATION OF AUXIN-BINDING SITES

hr at 27,000 rpm in the Spinco SW 27 rotor, then fractionatedand assayed as with other isopycnic gradients. A control regra-

dient was run in parallel, using another aliquot of particles fromthe high Mg2+ gradient which was diluted with gradient basalmedium containing 3 mM MgCl2 and recentrifuged into a secondhigh Mg2+ (3 mM) isopycnic gradient.

Binding Assays. Pellets that had been resuspended in standardbinding assay medium (24) were assayed for [14C]NAA-bindingas described previously (24), using an assay sample volume of 2ml; specific binding denotes the difference between the amountof 14C in duplicate pellets without, and with addition of, 10-4 MNAA (for details see ref. 24). NPA-binding was assayed in thesame manner, using 5 x 10-9 M [3H]NPA minus and plus 10-5 Munlabeled NPA.For assay of NAA-binding in density gradients, aliquots of the

fractions were diluted with an equal volume of 0.25 M sucrosecontaining 10-7 M [14CJNAA, 1 mM MgC92, and 20 mm Nacitrate-citric acid buffer (pH 5.3), which brought the pH of thetris-buffered gradient media to 5.5. Aliquots of this mixture (2ml each) were centrifuged without, and with addition of, 10-4 Munlabeled NAA. NPA-binding in gradient fractions was assayedsimilarly except that the particle suspension was diluted withthree times its volume of citrate-buffered assay medium in pre-paring the assay samples.Enzyme Assays. Cytochrome oxidase was determined by add-

ing an appropriate aliquot (10-50 ,lI) of particle suspension or

gradient fraction to 0.5 ml of 20 ,uM reduced Cyt c in 50 mm trisbuffer (pH 7.5), and following optical density at 550 nm with a

recording spectrophotometer. NADH:- and NADPH:Cyt c re-

ductases were assayed similarly but the medium contained 20,lM oxidized Cyt c, 80 ,mM NADH or NADPH, and 1 mm KCN.Glucan synthetases were determined by adding 100 ,u of

density gradient fraction to 40 ,l of 50 mm tris (pH 8) contain-ing 17.5 nCi of UDP-['4C]glucose (260 Ci/mol; from The Radi-ochemical Centre, Amersham, U.K.). For assay of glucan syn-thetase I, the samples also contained 58 mM MgCl2; for glucansynthetase II, they contained 0.5 mm unlabeled UDP-glucoseand no Mg2+. Samples were incubated 15 min at 25 C, then 1 mlof 70% ethanol was added, plus 50 ,l of 50 mm MgCI2 and 150,ul of boiled crude microsomal particles from maize (from 4 gfresh wt of coleoptile tissue/ml of suspension) in order to im-prove the recovery of labeled products. The mixture was imme-diately boiled for 1 min, and after standing overnight at 4 C, wascentrifuged 5 min at 1,000g. Precipitated particulate materialwas washed four times with 70% ethanol, which removed allunreacted radioactive substrate and ethanol-soluble by-prod-ucts, and was then suspended in scintillation fluid for determina-tion of radioactivity.

Particulate protein was determined by the method of Lowry etal. (16), after pelleting the particles by centrifugation; BSA was

used as standard. RNA was determined by the orcinol method(26).

Electron Microscopy. Gradient fractions were diluted withtwice their volume of 15% glutaraldehyde in 50 mm cacodylatebuffer (pH 7), and layered over 0.3 ml of 2% OS04 in the same

buffer containing 22% sucrose covered with a layer of 0.3 ml20% sucrose in the same buffer. These samples were centrifuged30 min at 45,000 rpm in the Spinco SW 56 rotor, depositing thefixed particles as a pellet at the bottom of the OS04 layer. Thesupernatant was drawn off, the pellet washed twice with icewater, then taken through an ethanol-water series into absoluteethanol, propylene oxide, and finally Epon by standard electronmicroscope preparatory procedures. Ultrathin sections were cutwith glass knives, stained with lead citrate, and examined with a

Hitachi 11E electron microscope.

RESULTS

Behavior in Differential Centrifugation. Exploratory experi-ments using differential centrifugation (Table I) showed thatNAA-binding activity is sedimented over a wide range of force xtime. The responsible particles are evidently quite heteroge-neous in size. However, the majority of the activity is "micro-somal"; the highest activity on a protein basis, and highest ratioof specific to nonspecific binding, is obtained from the particlessedimented at the highest force x time (Table I).NPA-binding activity similarly falls in a wide range of centrifu-

gal fractions (Table I), and by the usual criterion is predomi-nantly "microsomal." However, its distribution differs quantita-tively from that of NAA-binding activity, relatively more NPA-binding activity being sedimented at lower speeds. This suggeststhat NPA-binding sites may be located on different membranesthan those that bear NAA-binding sites, but it is clear that thetwo kinds of binding sites cannot be qualitatively separated bydifferential centrifugation.Complementary results can be obtained by rate-zonal centrif-

ugation (Fig. 1). Both NAA- and NPA-binding sediment slowlylike "microsomal" turbidity, but the distribution of NPA-bind-ing is shifted toward more rapid sedimentation rates comparedwith NAA-binding. The main peak of mitochondrial Cyt oxidaseactivity sediments more rapidly than the peaks of binding activity(Fig. 1), but part of the Cyt oxidase activity (which results givenbelow show is also due to mitochondria) sediments as slowly as

the microsomal material. Similar results are obtained by differ-ential centrifugation: about one-third of the mitochondrial activ-ity sediments with microsomal fractions and cannot be separatedfrom NAA- and NPA-binding activity by differential centrifuga-tion (data not presented).Behavior in Isopycnic Centrifugation. In order to obtain satis-

factorily sharp separations by isopycnic centrifugation, it was

Table I. Sedimentation of NAA- and NPA-binding Activities in Differential Centrifugation

Binding assays were performed on one-tenth of the particulate material of each centrifugal fraction, derived from10.8 gm fresh weight of coleoptile tissue, as described in Methods; 20,000 cpm of (14C)NAA or 5000 cpm of (3H)NPAwere supplied in each assay. Centrifugal force is given for rav.

Particulate Specific (14C)NAACentrifugation step protein (3H)NPA binding (14C)NAA binding binding per mg

per assay protein

force time Nonspecific Specific Nonspecific Specific

x g min mg cpm cpm cpm cpm cpm/mg

2,000 10 0.49 85 405 155 100 204

10,000 10 0.99 105 555 260 270 270

41,000 15 1.06 120 560 280 590 550

130,000 20 0.53 95 335 175 610 1,150

595Plant Physiol. Vol. 59, 1977

Plant Physiol. Vol. 59, 1977

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cles from a maize coleoptile homogenate. Sedimentation from left toright; bar on abscissa shows zone corresponding to the volume of homog-enate applied to the gradient. Homogenate was derived from 16 g freshwt of tissue and was centrifuged and assayed by procedures given under"Materials and Methods." Cyt oxidase determination used 1/45 of eachgradient fraction; NAA- and NPA-binding assays each used one quarter.

necessary to concentrate the membranous particles by centrifug-ing them onto a sucrose cushion rather than by pelleting them,presumably because during pelleting different types of mem-brane particles stick to one another. When this precaution istaken, the results shown in Figure 2A are obtained. NAA- andNPA-binding activity are largely separated into single peaks thatoccur at about 25% (w/w) sucrose (density 1.10 g cm-3) and38% sucrose (density 1.17 g cm-3), respectively. This provesthat most of these respective kinds of binding sites are indeedcarried by different membranes.Comparison ofNPA Binding with Marker Enzymes. The peak

of NPA-binding at 35 to 38% sucrose in isopycnic gradients(Fig. 2A) overlaps the peak of Cyt oxidase activity (Fig. 2B) at40% sucrose that is due to those mitochondria that are notremoved by the 6,800g precentrifugation. However, the peak ofNPA-binding is broader, and extends to lower densities. Elec-tron microscopic examination showed that fractions at 40 to41% sucrose, at the Cyt oxidase peak, contained almost exclu-sively mitochondria (micrographs not shown), whereas fractionsat 35 to 37% sucrose contained mitochondria plus smooth mem-brane vesicles (Fig. 3A). Despite the presence of mitochondriain the zone where NPA-binding is maximal, it is clear from thequantiiative differences in distributions of NPA-binding and ofCyt oxidase (Figs. 1 and 2) that NPA-binding sites are carried byparticles different from the mitochondria.The isopycnic distribution of NPA-binding activity generally

correlates with that of a low affinity, Mg2+-independent ,3-glucansynthetase activity which we call glucan synthetase II (Fig. 2C).Isopycnic centrifugation clearly separates the peak of glucansynthetase II activity from a sharp peak of Mg2+-dependent, highaffinity glucan synthetase activity which we call glucan synthe-tase I (Fig. 2C). Glucan synthetases I and II are similar to twotypes of glucan synthetase activities described previously fromAvena coleoptiles (28) and, like the Avena enzymes, form a

mixture of (3-1,3 and /8-1,4 linkages as seen by enzymic degrada-tion of their products. The properties of these synthetases will bepresented in detail elsewhere, but for present purposes, it suf-fices to note that glucan synthetase I closely resembles the Golgi-bound glucan synthetase described for other tissues (7, 25, 30)and its occurrence correlates with the presence of recognizableGolgi cisternae in the particle fractions. Glucan synthetase activ-ities that have properties and an isopycnic distribution similar toour glucan synthetase II have been held to be localized in plasmamembrane (7, 8, 10, 27, 30).

Comparison of NAA-binding with Markers. The sharp isopyc-nic peak of NAA-binding at about 25% sucrose (Fig. 2A)overlaps, but is clearly distinct from, a peak of yellow carotenoidpigment (Fig. 2D) at about 28% sucrose which probably showsthe distribution of proplastids (22). The distribution of NAA-binding conforms closely on the other hand with that ofNADPH:Cyt c reductase and of rotenone- and antimycin A-insensitive NADH:Cyt c reductase activities (Fig. 2B). These

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FIG. 2. Isopycnic density gradient fractionation of NAA- and NPA-binding activities and comparison enzymes associated with particles inthe 6,780g supematant of a maize coleoptile homogenate. Gradientcontained particles from 20 g fresh wt of tissue. In the binding assays(A), respectively 22,000 cpm of ['4C]NAA or 8800 cpm of [3HJNPAwere supplied; at the peak of their respective specific binding profiles,nonspecific binding (not shown) of NAA was 180 cpm and that of NPAwas 50 cpm. Assays for Cyt oxidase and NADH- and NADPH:Cyt creductases (B) used, respectively, 100, 8, and 100 ,ul of each gradientfraction out of a total volume of 3.5 ml. Assays for glucan synthetases Iand II (C; see "Materials and Methods") were done with 100 ,ul of eachfraction. Carotenoids (D) were determined on particles from 0.5 ml ofeach gradient fraction, as optical density at 455 nm obtained by extrac-tion with 1.5 ml of 7% acetone in methylene chloride. Separate experi-ments showed that the NADH:Cyt c reductase activity assayed in B iscompletely insensitive to antimycin A and to rotenone.

596 RAY

LOCALIZATION OF AUXIN-BINDING SITES

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FIG. 3. Electron micrographs of particulate fractions, from density gradients, as isolated for studies of NAA- and NPA-binding. (Bar equals 1gim.) A: Zone of peak NPA-binding from low Mg2+ gradient, 38% sucrose (density 1.17 g/cm3); B: zone of peak NAA-binding from similargradient, 25% sucrose (density 1.11 g/cm3); C: zone of peak NAA-binding from high Mg2+ gradient, 37% sucrose (density 1.17 g/cm3); D: NAA-binding particles obtained at 25% sucrose by treating a high Mg2+ preparation similar to C with EDTA and recentrifuging isopycnically as describedin the text.

enzymes are recognized as ER membrane markers (15). Theyband sharply at about 25% sucrose, as is characteristic of ERmembranes from other tissues when prepared in low Mg2+ mediathat strip ribosomes from the ER. The membrane material atthis peak consists (Fig. 3B) mainly of vesicular and cisternalmaterial suggestive of stripped RER (15).

Distribution of NAA-binding and Markers in High Mg2+Preparations. When prepared using homogenization and gra-dient media containing 3 to 4 mM Mg2+ ("high Mg2+"), toprevent stripping of ribosomes from the RER, the distribution ofNADH: and NADPH:Cyt c reductases are shifted to a broaderpeak at a much higher density (Fig. 4B), as was expected from

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FIG. 4. Isopycnic distribution of NAA- and NPA-binding activityand of comparison enzymes in a high Mg2+ preparation. Particles wereprepared from 25 g fresh wt of coleoptile tissue by procedure similar tothat of Figure 2 except that the homogenization and gradient mediacontained 4 mm and 3 mM MgCl2, respectively. Details of assays weresimilar to those listed for Figure 2.

experience with other plant tissues (15, 21). The distribution ofother enzyme activities and NPA-binding discussed above arerelatively little affected by high Mg2+ (Fig. 4). However, thedistribution of NAA-binding (Fig. 4A) is shifted in high Mg2+exactly like the ER marker enzymes. The new peak of NAA-binding activity, at about 35% sucrose, corresponds with thepeak of membrane-bound RNA (Fig. 5A). These facts indicatethat at least the great majority of the NAA-binding sites arelocated on RER membranes.

This was confirmed by "stripping" experiments in which mem-brane preparations made in high Mg2+ media were centrifugedisopycnically, the high density zone containing the peak ofNAA-binding and RNA (Fig. 5A) was treated with EDTA todissociate ribosomes from RER (15, 18), and the particles werethen recentrifuged isopycnically into a second gradient (Fig.5B).As a result of stripping, the distribution of both NAA-binding

and of NADH:Cyt c reductase was shifted to a much lowerdensity. Other membrane material in the original high densityzone was not conspicuously affected by stripping, leading to thelack of correspondence, in the second gradient (Fig. 5B), be-tween the peak of protein on the one hand and of Cyt c reductaseand NAA-binding activity on the other. The broader distributionof NAA-binding in Figure 5B compared with Figure 2A can beexplained by the fact that the stripping procedure did not removeall of the ribosomes from the RER membranes, as apparentfrom the data for particulate RNA in Figure SB.As a control for the stripping procedure, particles from the

RER region of a high Mg2+ gradient were diluted with gradientbasal medium containing 3 mM Mg2+ and recentrifuged into ahigh Mg2+ gradient. In this gradient, ER markers and NAA-

binding peaked again at 36.5% sucrose (data not shown), dem-onstrating that the density shift obtained in Figure SB was notdue to the recentrifugation itself, but to the treatment withEDTA which removed most of the RNA from these membranes.The peak fractions for NAA-binding from high Mg2+ gradients

consist, as anticipated from the foregoing, primarily of rough ERvesicles (Fig. 3C), plus some mitochondria and smooth mem-brane vesicles as expected from the overlap of distribution of ERWith that of mitochondria and of plasma membrane under highMg2+ conditions. When these particles were subjected to thestripping and regradient procedure just described, the particlesrecovered from the gradient fractions around 26% sucrosewhere NAA-binding and NADH:Cyt c reductase is now concen-trated consist of vesicular material lacking ribosomes but havinga frayed appearance suggestive of their origin by removal ofribosomes from RER (Fig. 3D).

DISCUSSION

The results show clearly that the principal NAA- and NPA-binding sites of maize coleoptile cells are located on differentcellular membranes. The NPA-binding membranes behave quite

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FIG. 5. Stripping of ribosomes from RER microsomes shifts thedensity of the membranes that bear NAA-binding sites. Two high Mg2"gradients similar to that of Figure 4 were prepared from a total of 38 gfresh wt of coleoptile tissue. Corresponding fractions were pooled andaliquots were used for the assays shown in A (above). Fractions compris-ing the indicated zone (30-37% sucrose) were pooled, treated withEDTA, and recentrifuged into a low Mg2+ gradient. The fractions fromthis gradient were assayed with results given in B (below).

598 RAY

-1

LOCALIZATION OF AUXIN-BINDING SITES

differently from ER or Golgi membranes as judged from experi-ence with other tissues. The isopycnic distribution of NPA bind-ing resembles closely that of non-Golgi glucan synthetase that isprobably plasma membrane-localized, and also resembles thedistribution inferred for plasma membranes of maize tissue bystudy of salt-stimulated ATPase (14). Therefore, as previouslyconcluded (8, 13), NPA-binding sites appear to be located atleast primarily on the plasma membrane. However, the availableevidence does not strictly exclude involvement of the tonoplast.The bulk of the NAA-binding sites are located on particles

that show characteristics distinctive of the RER, so we concludethat the RER is the principal locus of auxin-binding activity. Theauxin-binding sites carried by the RER are not located on theribosomes but on the RER membranes, since the binding capac-ity remains on the membranes after the ribosomes have beenstripped from them.

In low Mg2+ preparations (Fig. 2A), from the peak at 25%sucrose that represents sites carried by the ER, a tail of NAA-binding activity extends up to concentrations above 30% su-crose. In this region, the NAA-binding activity, although low, isgreater than expected in proportion to the very low activity ofthe ER marker NADH:Cyt c reductase (Fig. 2B). This feature,which has been observed consistently in gradients of the typeshown in Fig. 2, suggests that a small minority of the NAA-binding sites may be located on Golgi and/or plasma mem-branes. This agrees at least qualitatively with suggestions of Battand Venis (1); more definite indications favoring this conclusionwill be presented separately (Dohrmann and Hertel, in prepara-tion).The finding that auxin-binding sites are located principally on

ER membranes seems to conflict with the currently popular viewthat primary auxin action should be exerted at the plasma mem-brane (4, 9, 12, 17). One obvious possibility is that the auxin-binding sites located on the RER are not the physiological auxinreceptor sites but may have some other function, such as intra-cellular transport of auxin. While this cannot be conclusivelyrejected on the basis of available data, there is fairly suggestiveevidence that the major NAA-binding sites may in fact bephysiological auxin receptors (9; Ray et al., in preparation).

Therefore, consideration should be given to the possibilitythat primary auxin action actually occurs at ER membranes. Isuggest the following model for how auxin could bring about theH+ excretion that current evidence indicates is what actuallycauses accelerated cell enlargement (3, 11, 17). Combination ofauxin with its receptor sites on the ER could induce H+ transportfrom the cytoplasm into the ER cistemal space. The acid con-tained therein would be transported, along with secretory pro-teins contained in the ER space, to the cell exterior (cell wallspace), probably via the Golgi system. This model explains thewidely observed absolute latency of 10 to 20 min in auxin actionon elongation and on acid excretion (cf. refs 11, 29), sincetransport to the cell exterior via the Golgi system takes place ona time scale of this order (2, 6, 20; Ray et al., unpublished). Thepossibility of auxin-induced H+ excretion via vesicles has beensuggested already (3). If primary auxin action were exertedinstead on an H+ pump at the plasma membrane, as manycurrently assume, one would expect acid excretion and elonga-tion to respond almost immediately to presentation of auxin, asthey do in response to fusicoccin, which presumably acts at theplasma membrane (4, 17).The above model offers an explanation for the rapid blocking

of auxin-controlled proton excretion by inhibitors of proteinsynthesis (3, 17): secretory flow of products from the ER spaceoutward should come to a stop when secretory proteins are notbeing produced. The model may also explain why osmotic con-centrations of mannitol eliminate the auxin effect on H+ excre-tion (3). Osmotic water stress not only inhibits protein synthesis(5), but also halts deposition of cell wall polymers (23), indicat-ing that transport by the Golgi system is shut down.

599

The hypothesis of auxin action via receptors on the ER alsosuggests a possible connection between rapid and longer termeffects of auxin that have been held to represent separate actionsof the hormone (29). Longer term auxin effects that involvestimulation of protein synthesis might stem from improved deliv-ery of nascent secretory protein into the ER lumen, whichtransport might be coupled to the secretion of H+ across thesame membranes.

Acknowledgments - I thank R. Hertel and U. Dohrmann for their interest and advice and forhelp in some of the experiments, H. Grein for valuable technical assistance, and V. Young for theelectron microscopy.

LITERATURE CITED

1. BATT S, MA VENtS 1976 Separation and localization of two classes of auxin binding sites incorn coleoptile membranes. Planta 130: 15-21

2. BOWLES DJ, DH NORTHCOTE 1974 The amounts and rates of export of polysaccharidesfound within the membrane system of maize root cells. Biochem J 142: 139-144

3. CLELAND RE 1975 Auxin-induced hydrogen ion excretion: correlation with growth, andcontrol by external pH and water stress. Planta 127: 233-242

4. CLELAND RE 1976 Fusicoccin-induced growth and hydrogen ion excretion of Avena coleop-tiles: relation to auxin responses. Planta 128: 201-206

5. DHINDSA RS 1976 Water stress and protein synthesis. III. Subcellular distribution ofinhibition of protein synthesis. Z. Pflanzenphysiol 78: 82-84

6. GARDINER M, MJ CHRISPEELS 1975 Involvement of the Golgi apparatus in the synthesis andsecretion of hydroxyproline-rich cell wall glycoproteins. Plant Physiol 55: 536-541

7. HARDIN JW, JH CHERRY, DJ MORRE, CA LEMBI 1972 Enhancement of soybean RNApolymerase activity by a factor released by auxin from plasma membrane. Proc Nat AcadSci USA 69: 3146-3150

8. HARTMANN MA, G NORMAND, P BENVENISTE 1975 Sterol composition of plasma mem-brane enriched fractions from maize coleoptiles. Plant Sci Lett 5: 287-292

9. HERTEL R, KS THOMSON, VEA Russo 1972 In vitro auxin binding to particulate fractionsfrom corn coleoptiles. Planta 107: 325-340

10. HoDGES TK, RT LEONARD 1974 Purification of a plasma membrane-bound adenosinetriphosphatase from plant roots. Methods Enzymol 32: 392-406

11. JACOBS M, PM RAY 1976 Rapid auxin-induced decrease in free space pH and its relationshipto auxin-induced growth in maize and pea. Plant Physiol 58: 203-209

12. KASAMO K, T YAMAIa 1976 In vitro binding of IAA to plasma membrane-rich fractionscontaining Mg++-activated ATPase from mung bean hypocotyls. Plant Cell Physiol 17:149-164

13. LEMBI CA, DJ MORRE, KS THOMSON, R HERTEL 1971 1-N-naphthylphthalamic acid (NPA)binding activity of a plasma membrane-rich fraction from maize coleoptiles. Planta 99: 37-45

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Plant Physiol. Vol. 59, 1977