Proc. Natl Acad. Sci. USAVol. 78, No. 11, pp. 6608-6612, November 1981Biochemistry

Molecular basis of auxin-regulated extension growth androle of dextranase

(cell elongation growth/arabinan/dextran)

ANTON N. J. HEYNLouisiana State University, University of New Orleans, New Orleans, Louisiana 70122

Communicated by Michael Kasha, June 18, 1981

ABSTRACT The first step in the extension growth ofthe plantcell is a process in which the cell wall becomes ductile or plastic,after which the actual enlargement takes place passively under theinfluence of turgor. The nature of this process has not been ex-plained, although much research has been carried out concerningit. In the present report, it is shown that a specific enzyme, whichis identical or nearly so with dextranase (a-1,6-D-glucan 6-gluca-nohydrolase, EC 3.2.1.11) and is associated with the cell walls ofgrowing coleoptiles, plays a prominent role in this process. Theaction ofthis enzyme is dependent on the level ofgrowth hormone,auxin, in the tissue. Under its action, certain cell wall componentsare broken down to yield arabinose and glucose. These sugars arealso released during autolysis of cell wall material. The molecularlinkages broken in the process are probably the arabinogalactancrosslinks of the hemicellulose matrix, which are the main con-stituents of the wall containing arabinose. This is substantiated bythe finding that dextranase can break down arabinan and com-pounds containing arabinose chains with the release of arabinose,just as in the action of the enzyme on the wall. The breaking ofthese crosslinks will impart the necessary plasticity to the wall forcell extension to occur.

The essential step in auxin-regulated extension growth (elon-gation) of the plant cell is plasticization ("loosening") of the cellwall. Thereafter, the cell wall may be irreversibly extendedunder the influence of the turgor to bring about the actual en-largement of the cell (1-3). The nature of this physical modi-fication of the wall and the underlying process have remainedunknown until now.

In 1968, I undertook a study of the possible involvement ofenzymes in this process. Such involvement might be in agree-ment with findings that certain antibiotics inhibit cell elongationand plasticization of the cell wall (4-6) and that protein synthe-sis, as well as synthesis ofDNA and RNA, is a requirement forcell elongation (7-12). In that study, the presence of enzymesin elongating coleoptiles was investigated, and several glucan-ases were found to be associated with the cell walls (13).

In an effort to screen out the enzyme actually involved in theprocess of the modification of the cell wall under the influenceof the hormone, the effect of the hormone concentration in thetissue on the activity of the enzymes, at optimal pH range, wasstudied. The activity ofonly one glucanase was found to dependon the auxin level in the tissue and to be higher at higher auxinconcentration. This enzyme is dextranase (a-1,6-D-glucan 6-glucanohydrolase, EC 3.2. 1.11) (14, 15).

Whether this outstanding behavior is due to a special, directsensitivity of the enzyme to the hormone or to a more compli-cated situation such as the interplay of more than one enzymeneed not be considered at this point. The special behavior of

this enzyme, however, made it first choice for being investi-gated as the possible "master" enzyme in the process.The present report deals with the action ofdextranase in the

tissue and its possible role in the process ofauxin-regulated cellwall modification in coleoptiles of Avena.

MATERIALS AND METHODSThe plant materials were coleoptiles ofa pure strain, "Victory,"ofAvena sativa, grown in the dark for 4 days. Sections 1 cm longfrom the growing zone of 20 coleoptiles were ground or ho-mogenized, washed twice in water, and centrifuged at 2200x g. For hormone-rich tissue, the sections were taken from"normal" growing coleoptiles; for hormone-poor tissue, the sec-tions were taken from coleoptiles from which the hormone-pro-ducing tip had been removed for 21/2 hr ("decapitated" coleop-tiles), after which time the hormone content is known to be low.The assays for determining the enzyme activities, as de-

scribed (13), consisted of incubating the tissue pellets with0.25% solutions of specific substrates at 400C and pH 5.0 anddetermining the glucose or reducing substances (as glucoseequivalent released in the reaction mixture after 2-4 hr. Thevalues for the controls (substrate and pellet alone) were sub-tracted. For determining the action of the enzyme on the cellwall, the pellets were boiled for 10 min in water to destroy allinherent enzymes and incubated for 2, 4, or 20 hr at 40'C at pH5.0 with 3 or 4 ml of buffer solution containing (or not contain-ing) the enzyme. A few drops of toluene were added to preventmicrobial growth.The release of reducing substances and glucose in the reac-

tion mixture was determined by the ferricyanide method (16)and the glucose oxidase method,* respectively. The sugars inthe reaction mixture were identified after lyophilization by as-cending thin-layer chromatography on silicate plates with high-purity sugars as standards. The chromatograms were developedwith ethyl alcohol/pyridine/water, 3:1:1 (vol/vol), and treatedwith silver nitrate in acetone and alcoholic sodium hydroxide.A highly purified, uncontaminated, homogeneous dextran-

ase preparation was a gift from Merck, Sharp and Dohme (17);other dextranases were purchased from Worthington and Cal-biochem. a-1,3-Glucanase ("mutanase," Novo Industri, Den-mark) was a gift from E. T. Reese (U.S. Army Research Lab-oratories). Purified arabinan, purchased from Koch-Light(Bucks, England), had been prepared essentially by the methodof Hirst and Jones (18). A purified rapeseed arabinan was a giftfrom I. R. Siddiqui (Canadian Dept. of Agriculture, Ottawa)(19). High-purity sugars, arabinogalactan, and p-nitrophenyl-a-L-arabinofuranoside were purchased from Sigma. Dextrans ofvarious molecular weights were donated by R. L. Easterday(Pharmacia); samples of oligodextrans were donated by Kuni-

* Keston, A. S. (1956) Abstracts, 129th Meeting, American ChemicalSociety, Dallas, TX, April 8-13, p. 31C (abstr.).

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Proc. NatL Acad. Sci. USA 78 (1981) 6609

hiko Gekko (Faculty of Agriculture, Nagoya University, Japan)and of arabinogalactan and arabinoxylan, by H. Meier (Univer-sity of Freeburg, Switzerland).

RESULTSDextranase Activity Associated with Cell Wall Pellets. The

dextranase activity in cell wall pellets had originally been stud-ied (14, 15) with the viscometric method only. Those resultshave been substantiated and expanded by using other methodsto rule out any ambiguity (20) and to study the reaction produetsof this process. For studying dextranase activity of. the twogroups of tissue pellets, dextrans of low molecular weight (Mr998 and Mr 3000; Mn 2400-9521) proved to be the most suitablesubstrates. In all cases, the reducing power ofthe reaction mix-ture was larger at higher auxin levels than at lower levels (Table1). With Mr 998 dextran these values were not higher than thoseof the substrate alone. This points to a synthetic process thatis taking place at the same time as the cleavage process, theequilibrium between the two processes being regulated by thehormone (and the pH?). With the higher molecular weight dex-tran, the values were higher than those of the substrate.

Chromatographic data not only confirm the cleavage ofthesedextrans by the pellet but also show that the reaction productsare the same as those of the hydrolysis of these substrates bydextranase. The chromatogram in Fig. 1 Upper compares thebreakdown ofMr 998 dextran by the cell wall pellet (lanes fandg) with the hydrolysis ofthe same substrate by dextranase (lanes

h and i). In both cases, isomaltose, glucose, and probably someisomaltotriose are released. These substances are also formedin the autolysis of the pellet (lane e), but in much lowerconcentrations.

With dextran Mr 3000, only diffuse streaks with superim-posed spots ofisomaltose and isomaltotriose were obtained aftera 2½-hr incubation (Fig. 1 Lower). This can be explained by thelarger size of the dextran molecules.

Action of Dextranase on the Cell Wall Pellet. That the dex-tranase activity associated with coleoptiles plays a role in themodification ofthe cell wall in elongation may be inferred fromthe action of dextranase directly on the cell wall pellet. Reduc-ing substances and glucose were released under the influenceof the enzyme (Table 2). The amount of glucose released wasless than the glucose equivalent of the .reducing substances,perhaps because ofthe production ofsugars other than glucose.The chromatogram of these reaction products (Fig. 2) shows

that the cell wall material degrades markedly under the influ-ence of the enzyme, as indicated by the heavy streaks in lanesf, h, and j. With lower concentrations of the reaction mixture,the heavy streaks can be resolved into distinct, intense spots

Table 1. Dextranase action in cell wall pellets from high-auxinand low-auxin tissues on dextrans of low molecular weights

Substrate Auxin content. Reducing substances*Mr 998 High 0.638 0.356 0.945

Low 0.439 0.273 0.561None 0.418 1.106

Mr 3000 High 0.352 0.370Low 0.217 0.346None 0.195 0.282

Pellets from normal (high auxin) and decapitated (low auxin) co-leoptiles were incubated for 21/2 hr at pH 5.0 and 400C with 0.1% dex-tran (Mr 998 and 3000).*Reducing substances released, shown asmg of glucose-equivalent per20 sections.

a b c d e f g h i j k I

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FIG. 1. Thin.,layer chromatograms of products of the action of cellwall pellet and of dextranase (70 jug/ml) on 0.1% dextran in 3-hr in-cubation. (Upper) Mr 998 dextran. Lanes: a and j, glucose; b and k,isomaltose; c and 1, isomaltotriose; d, dextran; e, pellet alone; f and g,pellet with dextran; h, dextranase with dextran; i, as h, low concen-tration. (Lower) Mr 3000 dextran. Lanes: a, glucose; b, isomaltose; c,isomaltotriose; d, dextran; e and g, pellet and dextran; f and h, pelletalone; i, and j dextranase and dextran; k, glucose, isomaltose, andisomaltotriose.

corresponding to the loci of arabinose, glucose (lanes 1 and m),and, after 3 days' incubation, galactose (?) (lane n) and xylose.Indications of the presence of isomaltose are also seen. Whenwhole coleoptile sections were exposed to the action of the en-

Table 2. Action of dextranase on cell wall pelletsVariable* Enzyme Control DifferencesRed. sub. 0.561 0.191 0.370Glc 0.074 0.020 0.054

0.316

Red. sub. 0.375 0.073 0.302Glc 0.220 0.043 0.177

0.125

Pellets were incubated for 20 hr at pH 5.0 and 400C with 70 lAg ofenzyme.* Red. sub., reducing substances, as glucose equivalent in mg per 20sections; GIc, glucose released, in mg per 20 sections.

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Proc. Natl. Acad. Sci. USA 78 (1981)

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FIG. 2. Thin-layer chromatogram of products of the action of dex-tranase (70 ,ug/ml) on the cell wall pellet. Lanes: a, arabinose; b, glu-cose; c, galactose; d, xylose, arabinose, glucose, and galactose; e, pelletfrom normal coleoptiles; f, as e, with dextranase, 2-hr incubation; g,pellet from decapitated coleoptiles; h, as g, with dextranase, 2-hr in-cubation; i, pellet alone; j, pellet with dextranase, 72-hr incubation; k,xylose, arabinose, glucose, and galactose; 1-n, low concentrations ofdigest-l, pellet alone; m, pellet with dextranase, 2-hr incubation; n,pellet with dextranase, 72-hr incubation.

zyme, for a prolonged time (3-5 days), their tissues disinte-grated completely.The production of glucose and isomaltose is characteristic of

the hydrolysis of dextrans. The release of arabinose is a re-

markable feature (see Discussion) that is in agreement with theabove indications that another reducing sugar is produced inaddition to glucose during the action of dextranase on the wall.

Action ofDextranase on Arabinan and Arabinan-ContainingCompounds. To explain the presence of arabinose in the prod-ucts of the action of dextranase on the cell wall pellet, the pos-

sible hydrolysis of arabinan by this enzyme was investigated.Under the influence of dextranase, arabinan can be brokendown with the production of reducing substances (Table 3).The action by a-1,3-glucanase, which acts on the sidechains

of arabinan, is given for comparison, as well as the combinedaction by both enzymes on the same substrate.The chromatogram in Fig. 3 shows that, in this hydrolysis of

arabinan, increasing amounts of arabinose and oligomers are

released, as shown by the presence of arabinose spots of in-creasing intensities on the side ofstreaks over the entire lengthsof the lanes. After hydrolysis for a few days, only the arabinosespots remained, showing a complete breakdown ofthe arabinan.

Furthermore, it was found by thin-layer chromatography thatdextranase can also break down gum arabic, arabinogalactan,

Table 3. Action of a-1,6- and a-1,3-glucanase on arabinan

Enzyme Enzyme Control Difference

a-1,6 1.297 0.337 0.960a-1,3 0.792 0.337 0.455a-1,3 and

a-1,6 2.258 0.706 1.5522.117 0.706 1.411

Arabinan (0.1%) was incubated for 72 hr at pH 5.0 and 40'C withone enzyme (70,ug/ml) and 20 hr longer with the other enzyme added.Results are shown as reducing substances released as glucose equiv-alent in mg per 20 sections.

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FIG. 3. Thin-layer chromatogram of products of the action of dex-tranase (70 jug/ml) on 0.25% dialyzed arabinan. Lanes: a, xylose; b,arabinose; c, glucose; d, galactose; e, arabinan; f, arabinan with dex-tranase, 5 min; g, as f, 30 min; h, as f, 1 hr; i, as f, 3 hr; j, arabinose.

and arabinoxylan with the release of arabinose and oligosaccha-rides. p-Nitrophenyl a-L-arabinofuranoside was broken downwith the release of nitrophenol, which was determined colori-metrically at 400 nm.

Action of the Coleoptile Pellet on Arabinan. Because dex-tranase proved to be active on arabinan and because this enzymeis associated with coleoptile pellets, the ability of coleoptilepellets to act directly on arabinan was investigated. The chro-matogram in Fig. 4 shows that, under the influence of the pel-let, arabinan of high molecular weight was broken down intocomponents (oligomers) of lower molecular weight; this isshown by the streaks starting about halfway in lanes f-h. Thispattern is similar to that of the breakdown of high molecularweight dextrans by dextranase.

Self-Digestion (Autolysis) of the Cell Wall Pellet. To closethe circle ofenzyme activities studied in the process ofcell wallmodification, the direct action of the pellet on itself (autolysis)was considered. The controls ofthe pellet without enzyme (Fig.1 Upper, lane e; Fig. 2, lanes e, g, and 1) furnish chromato-graphic information about this. In Fig. 1, spots correspondingto glucose, isomaltose, and isomaltotriose were present after 3hr of autolysis. In Fig. 2, a strong spot corresponding to arabi-nose and weaker spots corresponding to glucose and probablyisomaltose were present after 72 hr (lane 1). After 2 hr of in-cubation, only streaks with maxima most likely of glucose andarabinose were seen (lanes e and g). The release of glucose inautolysis has been known for a long time (21); the release ofarabinose is a novel finding. In autolysis, therefore, patternsidentical to those of the digestion of the pellet by dextranaseoccur. This coincidence strongly suggests that the same enzy-matic process is involved in both cases and that the dextranaseaction normally occurs in growing coleoptiles.

DISCUSSIONThat certain glucanases (,&1,4- and ,B-1,3-glucanase and (3-glu-cosidase) would be involved in the process of extension growthhas- been suggested by several authors (22-28). Because none

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Proc. NatLAcad. Sci. USA 78 (1981) 6611

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FIG. 4. Thin-layer chromatogram of products of the action of thecell wall pellet on 0.25% arabinan. Lanes: a, xylose; b and i, arabinose;c, glucose; d, galactose; e, arabinan alone; f, arabinan with pellet fromnormal plants (high auxin); g, as f, with pellet from decapitated plants(low auxin); h, as f with boiled (3 min) pellet.

of these enzymes was found to exhibit, at optimal pH range, a

different activity at different auxin levels and growth rates,these enzymes are less likely to play a prominent role in theelongation of coleoptiles than the enzyme studied here, whichhad earlier been found to be "sensitive" to auxin (14, 15).

That this enzyme, dextranase, should play this role is sub-stantiated by the present experimental findings: an enzyme thatcan cleave dextrans, and therefore may be termed a "dextran-ase," is associated with growing cell walls; this enzyme has a

special sensitivity to auxin in that, at optimal pH range, its ac-

tivity depends on the auxin level in the tissue (being higher athigher auxin concentrations); dextranases are able to breakdown cell wall materials yielding products specific for dextran-ase activity and also arabinose; and the reaction products of au-tolysis ofthe cell wall are similar to those ofthe dextranase actionon the wall.The finding (29) that arabinose is released during the action

of dextranase on the wall, as well as during autolysis, appearsto be an important one which may lead to an answer to the ques-tion ofwhich cell wall components and chemical bonds are bro-ken in the modification of the wall in elongation.

In view ofthe structure ofarabinan (30-32) and modern con-

cepts ofenzyme specificity (33), it seems possible that dextran-ase is able to act on the a-1,5 linkages between the arabino-furanosyl rings in the backbone of arabinan; these haveessentially the same configuration as the a-1,6 linkages betweenthe glucopyranosyl rings in dextrans. The action of dextranase,which is an endoenzyme (at least when acting on dextran),would then be similar to that of the endoenzyme arabin(an)ase(34, 35), which preferentially cleaves the a-1,5 linkages in thebackbone. However, dextranase also exhibits some of the fea-tures ofthe exoenzyme arabinosidase (36-39) (which cleaves theterminal a-1,5 linkages of the backbone and the a-1,3 linkagesof the branches of arabinan) because it can cleave gum arabic,arabinogalactan, and nitrophenyl-a-L-arabinofuranoside butnot xyloglucan. It differs from both enzymes with regard to itspH optimum, 4.0-5.0 (41).

The release of arabinose from the cell wall under the actionof dextranase and the association of dextranase activity withgrowing coleoptiles suggests that, in the process of cell wallmodification, arabinose-containing compounds are acted uponby this enzyme. This points to the breakdown of certain com-

ponents of the hemicellulose matrix between the cellulose fi-brils in the wall.

Models of the hemicellulose in young cell walls have beenproposed (41) in which arabinogalactan chains would form thecrosslinks between rhamnogalacturonan and xyloglucan chainswhich are parallel to the cellulose fibrils in the cell wall and towhich the xyloglucan chains in turn are attached by hydrogenbonds. These arabinogalactan crosslinks may consist of shortarabinosyl chains (or blocks) alternating with short galactosylchains to form a heteropolymer chain (42). Because these het-eroarabans are the main arabinose-containing components ofthe wall, it is likely that the arabinose produced in the digestionof the wall originates from these polymer chains.

In an alternative model, it is proposed that the crosslinks ofthe wall consist of short arabinan chains continued by galactanchains which are attached as side branches to a hydroxyproline-rich protein ("extensin") taking the place ofrhamnogalacturonanin the previous model (43-45). Hydroxyproline arabinosideshave been isolated particularly from lower plants and this modelmay prevail in that group of plants. Dextranase can act equallywell on such crosslinks by cleaving their arabinan componentswith release again of arabinose.

In a third, older model, it is proposed that the wall is heldtogether by secondary, noncovalent interactions or entangle-ments between macromolecules (46). In that case, the actionby dextranase on either type of linkages of the arabinan com-

ponents would be effective by decreasing its degree of branch-ing and entanglement.

If these crosslinks were broken, the structural rigidity of thewall would be severed and the cellulose fibrils would be set free.Under stress, they could then slide along each other, and theductility of the wall would be changed so that it becomes plastic.The role ofdextranase in the plasticization ofthe wall could thusbe explained on the basis of the experimental findings in thisreport.The production of glucose in addition to arabinose could be

explained if it were assumed that short dextran stretches, even-

tually replacing the galactan stretches in the crosslinks (to forman arabinoglucan?), are present in coleoptiles and are brokendown by the dextranase. Other possibilities are not excluded,however.

Because only a few breaks, of some of the crosslinks, wouldhave a profound effect on the extensibility of the wall, the dex-tranase activity need not be great and no great release of re-action products, if any, can be expected under normal condi-tions of elongation. The disintegration of the tissue, observedunder prolonged action by the enzyme, however, can be ex-

plained by a complete breaking of all of these crosslinks.Moreover, it would be a requirement that this process is a

reversible one because otherwise no regulation would be pos-sible (2). The data with low molecular weight dextran in Table1 are believed to point to the existence of such a reverse syn-thetic process which can restore the breaks that have occurred.

In summary, the findings in this communication concerningthe presence and action of dextranase in growing coleoptilessuggests that this enzyme plays an important, if not essential,role in auxin-regulated extension growth. They may also con-tribute in revealing the molecular basis of this process.

I thank the Gulf South Research Institute (New Orleans, LA) for theuse of facilities, Drs. L. Chaiet and K. Nollstadt of the Merck, Sharp

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Proc. Natd Acad. Sci. USA 78 (1981)

and Dohme Research Laboratories (Rahway, NJ), Dr. E. T. Reese, U. S.Army Natick Research Laboratories, Dr. R. L. Easterday of PharmaciaFine Chemicals (Piscataway, NJ), Dr. K. Gekko, Faculty ofAgriculture(Nagoya University, Japan), Prof. H. Meier, University of Freiburg(Switzerland); Dr. I. R. Siddiqui, Canadian Dept. of Agriculture (Ot-tawa), and Dr. Darrel Wesenberg, U.S. Department of AgricultureExperimental Station (Aberdeen, OH) for samples and Prof. M. Kasha,Institute ofMolecular Biophysics (Florida State University, Tallahassee)for his encouragement in this work.

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