The Plant Cell, Vol. 1, 747-755, August 1989 O 1989 American Society of Plant Physiologists

Pectic Cell Wall Fragments Regulate Tobacco Thin-Cell- Layer Explant Morphogenesis

Stefan Eberhard, Nancy Doubrava, Victbria Marfa, Debra Mohnen,’ Audrey Southwick,* Alan Darvill, and Peter Albersheim

Complex Carbohydrate Research Center and Departments of Biochemistry and Botany, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602

Pectic fragments of cell wall polysaccharides, released from the walls of suspension-cultured sycamore cells by treatment with endopolygalacturonase, were tested for morphogenesis-regulating activity in a modified tobacco thin-cell-layer explant (TCL) bioassay (D. Mohnen, S. Eberhard, V. Marfa, N. Doubrava, P. Toubart, D. J. Gollin, T.A. Gruber, W. Nuri, P. Albersheim, and A. Darvill, manuscript submitted). The pectic fragments inhibited the formation of roots on TCLs grown on a root-inducing medium containing 15 micromolar indole-3-butyric acid and 0.5 micromolar kinetin. Addition of the pectic fragments to a root-inducing medium containing 7 micromolar indole-3-butyric acid and 0.15 micromolar kinetin caused roots to form on the basal end of TCLs. TCLs cultured on this medium in the absence of added pectic fragments formed roots along their entire length. The pectic fragments induced polar tissue enlargement and the formation of flowers on TCLs cultured on transition medium. The flower-inducing activity was stable to heat treatment and proteolytic digestion. Pectic fragments isolated from the walls of suspension- cultured tobacco cells were as effective as those from the walls of sycamore cells in inducing de novo flower formation in the TCLs. These results support the hypothesis that oligosaccharins from plant cell walls regulate morphogenesis.

INTRODUCTION

The plant cell wall is a dynamic structure that serves both structural and functional roles in plants (Albersheim et al., 1983). It has been shown that pectic polysaccharide frag- ments from the plant cell wall act as endogenous elicitors that induce plants to protect themselves against patho- gens keviewed by Ryan, 1987; Hahn et al., 1989). Frag- ments of plant cell wall polysaccharides have also been shown to regulate growth and development of plants. A fragment released from xyloglucan inhibits auxin-stimu- lated elongation of pea stem segments (York, Darvill, and Albersheim, 1984; McDougall and Fry, 1988, 1989). Frag- ments produced by partia1 acid hydrolysis of cell walls inhibit flowering and promote vegetative growth in Lemna gibba (Gollin, Darvill, and Albersheim, 1984). Organogen- esis of tobacco thin-cell-layer explants (TCLs) is altered by addition of cell wall fragments to the culture medium (Tran Thanh Van et al., 1985). Extracellular polysaccharides from soybean enhance cell expansion and separation when added to soybean callus tissue cultured on medium con- taining colchicine (Hayashi and Yoshida, 1989). These results suggest that the cell wall might be a repository of

’ To whom correspondence should be addressed. Current address: Department of Biological Sciences, Stanford

University, Stanford, CA 94305.

chemical signals that regulate plant development. Tran Thanh Van and colleagues (1985), in collaboration

with our laboratory, presented evidence that the addition of fragments from plant cell walls to TCLs cultured on various liquid media caused TCLs to form vegetative shoots rather than flowers or callus, and flowers rather than vegetative shoots. These observations stimulated us to attempt to use TCLs to study the role of plant cell wall fragments in plant morphogenesis. Our goals were to reproduce the experiments (Tran Thanh Van et al., 1985) that showed that plant cell wall fragments could alter TCL organogenesis, and to use the TCL bioassay in the purifi- cation and study of morphogenetically active cell wall fragments. However, in spite of extensive efforts to per- form the in vitro TCL assay as described (Tran Thanh Van et al., 1985), we were not able to obtain the defined TCL organogenesis. Therefore, experiments were undertaken to determine which factors had the greatest effects on morphogenesis in the tobacco TCL bioassay. These efforts led to the development of a modified thin-cell-layer bioas- say that is a sensitive and reproducible system for studies of plant morphogenesis (D. Mohnen, S. Eberhard, V. Marfa, N. Doubrava, P. Toubart, D.J. Gollin, T.A. Gruber, W. Nuri, P. Albersheim and A. Darvill, submitted).

The effects of endopolygalacturonase-released cell wall

748 The Plant Cell

fragments on morphogenesis in the modified TCL bioassay are presented in this paper. Enzyme-released fragments were used rather than fragments generated by partia1 acid- or base-catalyzed hydrolysis because enzyme-released wall fragments consist of a well-defined, well-characterized mixture of pectic oligo- and polysaccharides. The defined nature of the fragments will facilitate purification and char- acterization of morphogenetically active components. The results reported here confirm that pectic fragments can regulate tobacco thin-cell-layer morphogenesis. However, the specific changes in organogenesis induced by the pectic fragments are different from those previously reported.

RESULTS

Pectic Fragments of Cell Walls Alter TCL Morphogenesis

A large-scale preparation of endopolygalacturonase-re- leased fragments from suspension-cultured sycamore cell walls was used to determine which concentrations of indole-3-butyric acid (IBA) and kinetin in the media were optimum for the study of pectic fragment effects on TCL morphogenesis. The glycosyl composition of this pectic fragment preparation (EPGaA4), shown in Table 1, is sim- ilar to that of previous preparations of endopolygalacturon- ase-released pectic fragments (Darvill, McNeil, and Alber- sheim, 1978). Amino acids, if present, were below the limits of detection by gas chromatographic analysis of heptafluorobutyric anhydride amino acid derivatives (mod- ified method of MacKenzie and Tenaschuk, 1974). All results in this paper were obtained using EPGaA4 wall fragments. However, other identically prepared sycamore cell wall pectic fragments had similar effects on TCL morphogenesis.

The effects of pectic fragments on TCL morphogenesis were first examined by culturing TCLs on media containing a range of IBA and kinetin concentrations in the absence and presence of 10 pg/mL EPGaA4 wall fragments. The type and number of organs and the symmetry of tissue enlargement of TCLs cultured for 24 days to 25 days were determined by examining each organ of every TCL with a dissecting microscope. Representative TCLs were photo- graphed for a permanent visual record. Our scale for quantitating asymmetric (polar) enlargement of the TCLs is illustrated in Figure 1A. A value of O represents sym- metric enlargement, 1 represents asymmetric enlargement that results in a TCL of more or less triangular shape, and 2 represents polar enlargement. We have confirmed that polar enlargement occurs at the basal end of the TCL (D. Mohnen et al., submitted).

The addition of pectic fragments affected TCL morpho- genesis differently depending upon the concentrations of

Table 1. Glycosyl Composition of EPGaA4 Fragment Mixture" Glycosyl Residue Normalized

Arabinose Rhamnose Fucose Xylose Mannose Glucuronic acid Galacturonic acid Galactose

mo1 O/O

15.2 7.4 1.2 1.5 1.8 5.0 56.0 11.6

a Glycosyl residues <1 ?'O of total are not included in these data.

IBA and kinetin in the medium (Figure 1B). Pectic frag- ments changed the number of organs formed (+), in- creased the polar enlargement of the tissue (P), or had no effect on morphogenesis (-). A pectic fragment-induced change in the number of organs formed was accompanied, with one exception, by an increase in the polarity of tissue enlargement. In the exception, TCLs cultured on media containing a relatively high (15 pM) concentration of IBA showed little (<0.5), if any, polar tissue enlargement in response to pectic fragments, although the pectic frag- ments changed the number of roots formed. The most pronounced effects of endopolygalacturonase-released pectic fragments occurred on TCLs cultured on root-in- ducing and transition media (compare Figures 1 B and 1 C). TCLs cultured on transition media form either no organs or very few vegetative shoots, flowers, or roots (D. Moh- nen et al., submitted). Pectic fragments had little or no identifiable effect on TCLs cultured on flower-inducing and vegetative shoot-inducing media.

Severa1 changes were observed on TCLs cultured on root-inducing and transition media. Addition of pectic frag- ments to root-inducing and transition media generally caused an increase in asymmetric (polar) enlargement of the TCLs. In some media, the change in polarity was accompanied by a change in the number, type, or distri- bution of organs. Four media were used for further analysis of the pectic fragment effects. The hormone concentra- tions of these media and the effects on TCL morphoge- nesis of the addition of pectic fragments at a concentration of 10 pg/mL are summarized in Table 2 and discussed in more detail later in the paper. Photographs of representa- tive TCLs cultured on these media, both with and without fragments, are presented in Figure 2.

Pectic Cell Wall Fragments Alter Morphogenesis of TCLs Cultured on Root-lnducing Medium

Addition of pectic fragments to a root-inducing medium containing 15 pM IBA and 0.5 pM kinetin resulted in

Pectic Fragments and TCL Morphogenesis 749

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Figure 1. Overview of Morphogenesis of TCL Explants on Media with and without Pectic Fragments.

(A) Scale used for the quantitation of asymmetric (polar) TCL tissue enlargement. (B) Effect of the addition of pectic fragments to various culture media on TCL morphogenesis. + indicates a difference of >2 organs per TCL in addition to an increased polar tissue enlarge- ment of > O 5 [see (A)]. The one exception was TCLs cultured on a medium containing 15 pM IBA and 0.5 pM kinetin in which polar tissue enlargement was < O 5 P indicates a mean quantitative change in polarity of > O 5 [see (A)], but with a difference of (2 organs per TCL. - indicates no significant effect of adding cell wall fragments. (C) Schematic representation of the ranges of IBA and kinetin concentrations (zones) in media without cell wall fragments that induce the formation of roots, vegetative shoots, or flowers in TCLs (D. Mohnen et al., submitted). R , root zone; F, flower zone; V, vegetative shoot zone; T, transition zone. [Note: (B) and (C) are not drawn to scale.]

Table 2. Effect on Organogenesis and Polarity of TCLs of Adding 1 O pg/mL Pectic Cell Wall Fragments to Media Containing the lndicated IBA and Kinetin Concentrations

Effect of Adding Pectic Fragments IBA Kinetin

PM PM lnhibition of root formation 8-15 0.25-0.5 lnduction of polar root formation 7-8 0.15 lnduction of polar tissue enlargement 4-6 0.5 lnduction of de novo flower formation 1.5-2 0.75-0.9

inhibition of root formation on TCLs. lncreasing the con- centration of pectic fragments from 0.1 pg/mL to 10 pg/ mL resulted in increasing inhibition of root formation, as shown in Figure 3. lnhibition of root formation on TCLs cultured on 8 pM IBA and 0.3 pM kinetin was accompanied by a decrease in the size of the TCL and a more pro- nounced polarity of tissue enlargement (see Figure 2A).

Pectic fragments added to a root-inducing medium that contained 7 pM IBA and 0.15 pM kinetin changed the location, but not the number of roots formed on the TCLs (see Figure 2B). In this medium, pectic fragments at a concentration of 1 O pg/mL caused approximately 90% of the roots to form at the basal end of the TCLs. Only approximately 25% of the roots formed on the basal end of control TCLs cultured on the same medium without pectic fragments; that is, the roots were located over the entire length of the TCL. The polar formation of roots in the presence of pectic fragments was accompanied by polar enlargement of the TCLs.

Pectic Cell Wall Fragments Alter Morphogenesis of TCLs Cultured on Transition Medium

TCLs incubated on a transition medium containing 4 pM IBA and 0.5 pM kinetin formed no organs or, on average, less than one flower, root, or vegetative shoot per TCL. Addition of pectic fragments at a concentration of 1 O pg/ mL to this transition medium caused tissue enlargement at the basal end of the TCLs. The addition of pectic fragments at a concentration of 1 O pg/mL completely inhibited any roots that would have otherwise formed on TCLs cultured on this medium (see Figure 2C). We occa- sionally observed a concomitant induction of a few flowers or vegetative shoots.

The formation of flowers was observed when pectic fragments were added to a transition medium that con- tained 1.5 pM IBA and 0.9 pM kinetin. Addition of pectic fragments at a concentration of 0.1 pg/mL to 10 pg/mL resulted in a concentration-dependent increase in the num- ber of flowers formed per TCL, as shown in Figure 4.

750 The Plant Cell

Figure 2. Effects of Pectic Fragments on TCL Morphogenesis.

Each photograph shows a control TCL (left) and a TCL cultured on the same medium supplemented with 10 ng/mL EPGaA4 (right).(A) Inhibition of root formation on a medium containing 8 /uM IBA and 0.3 ,uM kinetin.(B) Induction of the polar formation of roots on a medium containing 7 /^M IBA and 0.15 ̂ M kinetin.(C) Inhibition of roots and induction of flowers or vegetative shoots on a medium containing 4 ^M IBA and 0.5 /iM kinetin.(D) Induction of flower formation on a medium containing 1.5 /*M IBA and 0.8 uM kinetin.

Partial Characterization of the Pectic Cell WallFragments That Exhibit Morphogenesis-AlteringActivity

The results described above demonstrated that additionof pectic fragments to the culture medium had four differ-ent effects on TCL morphogenesis, and that each effectdepended upon the concentrations of auxin and cytokininin the medium (Figure 2). The EPGaA4 pectic fragmentswere prepared by treatment of sycamore cell walls withan apparently homogeneous endopolygalacturonase puri-fied from the culture medium of the fungus Aspergillusniger. The possibility that the morphogenesis-altering ac-tivity was due to a component inadvertently introducedwith the Aspergillus enzyme was tested by treatment of

sycamore cell walls with endopolygalacturonases isolatedand purified from two other fungi, Rhizopus arrhizus andColletotrichum lindemuthianum. Three pectic fragmentpreparations of sycamore cell walls were obtained bytreating separate cell wall samples with the three fungalendopolygalacturonases. Each of the three pectic frag-ment preparations caused the inhibition of root formationon TCLs cultured on a root-inducing medium and theinhibition of root formation, induction of polar growth, andoccasional induction of a few flowers or vegetative shootson TCLs cultured on a transition medium (4 /^M IBA and0.5 ijM kinetin). These results are consistent with thehypothesis that pectic oligo- or polysaccharides in EPGaA4are responsible for the morphogenesis-altering activities.

We could not rule out that very small amounts of protein

Pectic Fragments and TCL Morphogenesis 751

6 , I IBA (15 pM)

Kinetin (0.5 pM)

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O 1 2 3 4 5 6 7 8 9 10

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Figure 3. lnhibition of Root Formation by Pectic Fragments.

Mean numbers (+SE) of roots formed per TCL after 24 days to 25 days of culture on a root-inducing medium (15 pM IBA and 0.5 pM kinetin) containing the indicated amounts of EPGaA4 pectic fragrnents. Data represent four experirnents with a minimum of eight replicates per experiment.

(below detection limits) were present in the EPGaA4 frag- ments. Therefore, we investigated the possibility that the morphogenesis-altering activity resided in a protein or heat-sensitive enzyme by incubating EPGaA4 fragments for 1 hr at 37OC with sufficient amounts of a nonspecific protease (Pronase) to degrade a protein sample of a mass equivalent to the total mass of the pectic fragment sample. The fragments were then heated at 120°C for 10 min to inactivate the protease. Other EPGaA4 fragments were subjected only to the heat treatment. The flower-inducing activity of the pectic fragments was stable to both the heat treatment and the proteolytic digestion, as illustrated in Figure 5. Heat-inactivated protease did not induce flower formation, nor did a mixture of monosaccharides equiva- lent to the molar glycosyl composition of EPGaA4 (see Table 1 and Figure 5). These results supported our hy- pothesis that the active component in EPGaA4 was a pectic oligo- or polysaccharide of cell wall origin.

Light causes a degradation of IBA in the TCL bioassay medium (D. Mohnen et al., submitted). Measurement of the amount of IBA remaining in medium containing 6 pM IBA and 0.5 gM kinetin incubated for 12 days under 55 pE m-2 sec-’ light, with and without pectic fragments at a concentration of 1 O gg/mL, showed that pectic fragments had no effect on IBA stability in the medium. 60th with and without fragments, 80% of the IBA added to the medium was degraded. Also, equilibrium dialysis indicated that no detectable binding occurred between the pectic fragments and IBA in the medium (data not presented).

Altering the pH of the culture medium between 3.8 and 6.15 changed the number of organs formed on the TCLs (D. Mohnen et al., submitted). Adding pectic fragments to the TCL bioassay medium caused no significant change in the pH of the medium (<0.05 pH units).

Pectic Fragments of Tobacco Cell Walls lnduce the Formation of Flowers on TCLs

The results described above established that pectic frag- ments isolated from the walls of suspension-cultured syc- amore cells were able to alter morphogenesis of tobacco explants. If cell wall fragments serve as regulators of morphogenesis in vivo, then fragments of tobacco cell walls should also be able to regulate morphogenesis in tobacco explants. To examine this question, pectic frag- ments were isolated from the walls of suspension-cultured tobacco cells derived from the same variety of tobacco as that used in the TCL bioassay. The induction by pectic fragments from tobacco cell walls of de novo flower for- mation on TCLs cultured on 1.5 pM IBA and 0.8 hM kinetin, shown in Figure 6B, is comparable to the effect Of pectic fragments from sycamore cell walls shown in Figure 6A. The variation between the numbers of flowers induced by sycamore and tobacco pectic fragments is no greater than that observed between different preparations of sycamore wall pectic fragments (S. Eberhard, N. Doubrava, V. Marfa, D. Mohnen, A. Southwick, A. Darvill, and P. Albersheim, unpublished data).

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Figure 4. lnduction of Flower Formation by Pectic Fragments.

Mean numbers (*SE) of flowers formed per TCL after 24 days to 25 days of culture on a transition medium (1.5 LLM IBA and 0.9 pM kinetin) containing the indicated amount of EPGaA4 pectic fragments. Data represent four experiments with a minimum of five replicates per experiment.

752 The Plant Cell

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tissue enlargement. Thus, the additiorr of pectic fragments to the culture medium has a variety of effects on TCL morphogenesis, depending upon the auxin and cytokinin concentrations in the medium.

We demonstrated that pectic fragments can induce the de novo formation of flowers on TCLs from floral branches. Tissues of floral branches and stem segments from the flowering part of the plant are more readily induced to flower than tissues from the vegetative parts of the plant (Chouard and Aghion, 1961; Wardell and Skoog, 1969; Tran Thanh Van, 1973; Hillson and LaMotte, 1977). We

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Biologically active carbohydrate molecules from funga1 and plant cell walls (oligosaccharins) can serve as signal mol- ecules in plants (Albersheim et al., 1983). Our results demonstrate that pectic cell wall fragments isolated from sycamore and tobacco suspension-cultured cells regulate tobacco TCL morphogenesis. The most dramatic effect of the pectic fragments occurs when they are added to transition media (1.5 pM to 2 pM IBA, 0.75 pM to 0.9 pM kinetin) in which the fragments induce the formation of flowers on TCLs. Pectic fragments at a concentration of 1 O pg/mL inhibit formation of roots on TCLs when added to root-inducing media containing 8 pM to 15 pM IBA and 0.25 pM to 0.5 pM kinetin. The same concentration of pectic fragments, when added to a root-inducing medium containing lower levels of auxin and cytokinin (7 pM IBA and 0.15 pM kinetin), cause the position of roots formed on TCLs to change from an even distribution over the TCL surface to an asymmetric distribution on the basal end of the TCL. Addition of pectic fragments to a transition me- dium containing 4 pM IBA and 0.5 pM kinetin causes polar

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Pectic Fragments and TCL Morphogenesis 753

do not know whether pectic fragments can induce flowers on TCLs from vegetative tissues.

The pectic fragment-induced changes in organogenesis described in this paper cannot be directly compared to those reported previously (Tran Thanh Van et ai., 1985) since the culture conditions have been modified (D. Moh- nen et al., submitted). Using the conditions described here, we did not observe a change from vegetative shoots to flowers or a change from flowers to vegetative shoots, as previously described. However, we occasionally observed a change from no organs to the formation of a few (0.5 to 2) vegetative shoots when TCLs were cultured on transi- tion medium containing 4 pM IBA and 0.5 pM kinetin. This might be qualitatively similar to the change from callus to vegetative shoots observed by Tran Thanh Van et al. (1 985).

A goal of these studies is to elucidate the mechanism by which pectic fragments induce the observed changes in morphogenesis. In comparing the data on cell wall fragment-induced changes in TCL morphogenesis with the types and numbers of organs produced on TCLs cultured on a range of IBA and kinetin concentrations (D. Mohnen et ai., submitted), we found a similarity between the effects of adding pectic fragments and the effects of reducing the concentration of IBA in the culture medium. For example, pectic fragments at a concentration of 10 pg/mL added to a medium containing 7 pM IBA and 0.1 5 pM kinetin induced polar formation of roots on TCLs that otherwise produced roots over the entire surfaces of the TCLs. TCLs grown on a medium containing a lower IBA concentration (4 pM IBA rather than 7 pM IBA) produced roots in a polar manner even when no pectic fragments were added (D. Mohnen et al., submitted). However, the similarity between the effects of adding pectic fragments and of reducing IBA in the medium is not reciprocal. For example, pectic frag- ments have no effect on the number of flowers formed on TCLs cultured on a flower-inducing medium (0.5 pM IBA and 0.5 pM kinetin). Yet incubation of TCLs on media containing reduced levels of IBA (e.g., 0.1 pM or 0.2 pM) results in a reduction in the number of flowers (D. Mohnen et ai., submitted). Thus, the actual mechanisms by which pectic fragments effect TCL explant morphogenesis can- not be explained merely by the increase or decrease in concentrations of auxin or cytokinin available to the TCLs. This mechanism, as those of auxin and cytokinin, remains to be elucidated.

Two other examples of regulation of plant morphoge- nesis by cell wall fragments have been described. York, Darvill, and Albersheim (1 984) demonstrated that base- solubilized sycamore cell wall fragments could inhibit auxin-induced elongation in pea stem segments. The ac- tive component has been shown to be an oligosaccharide fragment of xyloglucan (McDougall and Fry, 1988, 1989), a hemicellulosic polysaccharide present in the primary cell walls of higher plants. Gollin, Darvill, and Albersheim (1 984) inhibited flowering and promoted vegetative growth in L.

gibba by the addition to the culture medium of cell wall fragments released from sycamore cell walls by partia1 acid hydrolysis. Since acid-released cell wall fragments consist of a mixture of both hemicellulosic and pectic fragments, it is possible that the active component in the EPGaA4 fragments used in the present studies is similar to that used by Gollin, Darvill, and Albersheim (1 984).

Based on the earlier studies and the work reported in this paper, we hypothesize that pectic fragments play a role in plant morphogenesis. To test this hypothesis, the active component(s) in the EPGaA4 pectic fragment mix- ture must be identified. The evidence that the biologically active component(s) is a carbohydrate is strong. The active fragments originate from well-washed plant cell walls (which are 90% carbohydrate) and are released from the walls by an enzyme that cleaves wall polysaccharides. The pectic mixture released by endopolygalacturonase (e.g., the EPGaA4 fragments) is known to be composed primar- ily of three types of molecules: rhamnogalacturonan I, rhamnogalacturonan II, and oligomers of galacturonic acid (York et ai., 1985). The biologically active molecules chro- matograph as large molecules in association with the endopolygalacturonase-released wall fragments. The ac- tive component(s) is stable to mild acid and base treat- ments. Moreover, the biological activity of the wall frag- ments is destroyed by a homogeneous endoglycanase (V. Marfa, D. J. Gollin, S. Eberhard, D. Mohnen, A. Darvill, and P. Albersheim, unpublished results). If the active moiety is a carbohydrate, depending upon its nature and size, the concentration at which the carbohydrate effects morpho- genesis is between 10-6 M and IO-' M. If the active substance is an undetected, quantitatively minor constit- uent of the wall fragments, it would have to be active at <I O-' M. Purification of the morphogenetically active com- ponent(s) in the pectic cell wall fragment mixture is in progress.

METHODS

Plant Material

Nicotiana tabacum L. cv Samsun line 5 plants (Tran Thanh Van et al., 1985) were grown in a greenhouse at 23OC to 31°C. Supplemental lighting was provided by high-pressure sodium lights to maintain a 14-hr day length. Plants were grown in a peat- lite soil mix (Fafard No. 3, Conrad Fafard, Springfield, MA) in 20- cm clay pots and fertilized twice each week with Peters 20-20-20 fertilizer (containing 473 ppm nitrogen). Since arnbient ozone levels were too high for growth of healthy tobacco plants in the greenhouse from late spring to early autumn, the air entering the greenhouse was passed through carbon filters (RSE, New Balti- more, MI) that removed approximately 80% of the naturally oc- curring ozone from the air.

754 The Plant Cell

Thin-Cell-Layer Bioassay

The TCL morphogenesis bioassay is a modification (D. Mohnen et al., submitted) of an assay developed previously (Tran Thanh Van, Thi Dien, and Chlyah, 1974; Tran Thanh Van et al., 1985). The second and third primary floral branches of a typical tobacco inflorescence were cut into 7-cm to 8-cm sections when approxi- mately 30% of the flowers had produced green fruits. Floral branches were soaked 2 min in 0.5% Tween 20, surface-sterilized 8 min in 10% commercial bleach (Clorox containing 5.25% NaOCI), and rinsed three times (total of 1 O min) in sterile deionized water. Strips of tissue, approximately 1 mm wide and composed of one layer of epidermal cells, 2 to 3 layers of chlorenchyma, and 3 to 6 layers of collenchyma and/or parenchyma, were cut from the floral branch tissue. TCLs approximately 1 O mm long were cut from the tissue strips and floated individually on 2 mL of a Linsmaier and Skoog (1965) medium containing 167 mM glucose (basal medium) and the indicated amounts of indolebutyric acid and kinetin (Sigma). The media were filter-sterilized using 0.2-pm filtration units (Nalgene Labware, Rochester, NY). The pH of each medium was adjusted to 5.8 by titration with KOH. The media and individ- ual TCLs were placed in 7-mL wells of 12-well cell culture cluster dishes (GIBCO), and the dishes were sealed with two layers of Parafilm (American Can Co., Greenwich, CT). The TCLs were incubated at 24OC under cool-white fluorescent lights (Sylvania, Danvers, MA) at 55 ? 5 pE m-* sec-'. After 23 days to 25 days of culture, the type of organogenesis of the TCLs was visually scored using a dissecting microscope to examine each organ. The three types of organs (roots, flowers, and vegetative shoots), the general size, and the degree of polar enlargement of the TCLs were recorded. The root(s), vegetative shoot(s), or flower(s) that grew from a single initial shoot or root meristem was defined as one organ. Thus, an individual flower that formed directly on the TCL surface would be scored as one flower, as would an inflo- rescence having five flowers. Likewise, a primary root with three lateral roots would be scored as one root. When pectic fragments were tested, a stock solution was filter-sterilized using a 0.2-pm nylon membrane syringe filter (Nalgene) and aseptically added to the medium.

Plant Cell Wall lsolation

Cultures of sycamore (Acer pseudoplatanus) cells, originally iso- lated by D.T.A. Lamport in 1958, have been maintained in the dark in our laboratory since 1960. The cells are grown on the modified M-6 medium (Torrey and Shigemura, 1957) and subcul- tured into fresh medium every 7 days. Primary cell walls of suspension-cultured sycamore cells were isolated as described (Talmadge et al., 1973).

A tobacco (N. tabacum L. cv Samsun) suspension culture was established in our laboratory from freshly isolated pith callus tissue. The cultured cells were grown in the dark on an LS medium (Linsmaier and Skoog, 1965) supplemented with 1 mg/L 2,4- dichlorophenoxyacetic acid and 30 g/L sucrose. The cells were subcultured to fresh medium every 14 days.

Purification of Endopolygalacturonase

Endo-oc-l,4-polygalacturonase was purified to homogeneity from a commercial preparation of Aspergillus niger pectinase by car-

boxymethylcellulose chromatography, preparative isoelectric fo- cusing, and gel-permeation chromatography on Sephadex G-50, as described (Cervone et al., 1987).

Comparative studies were done using an endo-a-l,4-polyga- lacturonase from Colletotrichum lindemuthianum prepared as de- scribed (English et al., 1972; York et al., 1985), and an endo-a- 1,4-polygaIacturonase from Rhizopus arrhizus purified from a commercial preparation of pectinase (Sigma No. P2401) by a modification of the method of Lee and West (1981).

lsolation of Pectic Cell Wall Fragments

Pectic polysaccharides were extracted from purified walls of suspension-cultured sycamore and tobacco cells by digestion of the cell walls with endo-~-l,4-polygalacturonase from A. niger as described by York et al. (1985).

Glycosyl Composition Analysis of Pectic Fragments

Glycosyluronic acid and neutra1 glycosyl residues of EPGaA4 pectic fragments were simultaneously quantitated as their per-0- trimethylsilyl methyl glycosides, prepared and analyzed as de- scribed (Chambers and Clamp, 1971; York et al., 1985).

Treatment of Pectic Fragments with Protease

EPGaA4 pectic fragments (1 mg in 1 mL H20) were incubated with 1 unit Pronase (Boehringer Mannheim) for 1 hr at 37°C and heated at 120°C for 10 min to destroy enzyme activity. The proteolytic activity of the Pronase-cell wall fragment solution, both before and after the 120°C treatment, was measured as Azocoll- digesting activity (Ragster and Chrispeels, 1979). A 50-pL aliquot of the Pronase-treated cell wall fragment solution was incubated with 15 mg of Azocoll (Behring Diagnostics) in a total volume of 1.5 mL for 1 hr at 37°C followed by incubation at 4OC for 15 min. The reaction mixture was then centrifuged for 5 min at 15,6009, and the absorbance at 520 nm was recorded.

We thank our colleagues at the CCRC for their help and valuable discussions, Laura Kiefer for purification of endo-a-1 ,4-polygalac- turonase from R. arrhizus, Alan Koller for the amino acid analysis of the pectic fragments, and Andy Tull and Donna Jarnagin for their assistance in growing the tobacco plants. This work was supported in part by grant DE-FG09-85ER13425 from the U.S. Department of Energy (DOE), and by DOE grant DE-FGO9- 87ER1381 O as part of the U.S. Department of Agriculture/DOE/ National Science Foundation Plant Science Centers program.

Received May 12, 1989; revised June 19, 1989.

Pectic Fragments and TCL Morphogenesis 755

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DOI 10.1105/tpc.1.8.747 1989;1;747-755Plant Cell

S. Eberhard, N. Doubrava, V. Marfa, D. Mohnen, A. Southwick, A. Darvill and P. AlbersheimPectic Cell Wall Fragments Regulate Tobacco Thin-Cell-Layer Explant Morphogenesis.

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