P-Glucan Synthesis in the Cotton Fiberâ€™

Plant Physiol. (1993) 101: 1149-1156

P-Glucan Synthesis in the Cotton Fiber’

111. ldentification of UDP-Glucose-Binding Subunits of P-Glucan Synthases by Photoaffinity Labeling with [P-32P]5’-N3-UDP-Glucose

Likun Li, Richard R. Drake, Jr.*, Sarah Clement3, and R. Malcolm Brown, Ir.*

Department of Botany, The University of Texas at Austin, Austin, Texas 7871 3-7640

Using differential product entrapment and photolabeling under specifying conditions, we identified a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Cossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring j3-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of &1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring &1,3-glucan synthesis, is coincident with B- 1,3-glucan synthase activity, and is calcium dependent. The possi- ble involvement of other polypeptides in the synthesis of &1,3- glucan is discussed.

Cellulose is a fundamental component of the cell walls of higher plants, but the process of cellulose biosynthesis has remained one of the major unresolved problems in plant biology (Read and Delmer, 1991). Although the catalytic subunit of cellulose synthase in Acetobacter xylinum was identified by Lin et al. (1990), the efforts to identify cellulose synthase of higher plants have been seriously hampered by the inability to detect enzyme activity in cell-free systems. Therefore, much attention has been focused on the identification and purification of another glucan synthase, namely, callose synthase (Read and Delmer, 1987; Delmer and Stone, 1988; Fink et al., 1990; Frost et al., 1990; Mason et al., 1990; Dhugga and Ray 1991a, 1991b). These efforts have been focused not only on the physiological functions of this enzyme (Delmer, 1987; Kauss, 1987; Delmer and Stone, 1988) but also on the question of whether callose synthase and cellulose synthase are part of the same enzyme complex (Jacobs and Northcote, 1985; Delmer, 1987).

Recently, severa1 reports have suggested that the cellulose

’ This work was supported by National Science Foundation grant DCB 8903685 to R.M.B.

* Present address: Department of Biochemistry and Molecular Biology, The University of Arkansas for Medica1 Science, 4301 West Markham Street, Mailslot 515, Little Rock, AR 72205.

Present address: Newnham College, Cambridge University, Cambridge CB 39DF, England.

* Corresponding author; fax 1-512-471-3573. 1149

synthase of higher plants may have homology with A. xylinum cellulose synthase (Amor et al., 1991; Mayer et al., 1991). An antibody derived from a genetically cloned A. xylinum cellulose synthase has been used as a probe, and the occurrence of immunologically cross-reacting proteins in higher plants has been found (Mayer et al., 1991). Further- more, 32P-cyclic diguanylic acid has been used as another probe and cyclic diguanylic acid-binding polypeptides in cotton have been found (Amor et al., 1991).

We reported (Li and Brown, 1993; Okuda et al., 1993) that a certain quantity of ANIP (about 4% of the total glucans), containing exclusively @-1,4-glucan, could be synthesized in vitro with an optimal combination of cellobiose, c-3’:5’-GMP (or cyclic diguanylic acid), magnesium, calcium, and digitonin, whereas P-1,S-glucan (callose) could be synthesized with only cellobiose and calcium as effectors. Based on the fact that different products can be synthesized under specifically defined and repeatable conditions, we have developed a protocol to differentially entrap and photolabel both glucan synthases. This method permits enrichment of the particular enzyme based on optimal conditions for the synthesis of its product. This technique also allows photolabeling of the enzyme to ascertain which enzyme was differentially entrapped under conditions optimal for the specific product. Obviously, the putative catalytic subunit for cellulose synthase should meet the following criteria: (a) it should be coincident with the synthesis of ANIP, (b) it should be present in the protein fraction differentially entrapped under conditions of F1,4, and (c) it should be magnesium dependent. The putative catalytic subunit for callose synthase should meet the following criteria: (a) it should be coincident with the synthesis of @-1,3-glucan, (b) it should be present in the protein fraction differentially entrapped under conditions of F1,3, and (c) it should be calcium dependent.

Abbreviations: AN reagent, acetic/nitric acid reagent; ANIP, acetic/nitric acid reagent-insoluble product; c-3’:5’-GMP, cyclic 3’:5’- GMP; CBH I, cellobiohydrolase I; EPF1.3, entrapped proteins obtained under conditions favoring P-1,3-glucan synthesis; EPF1,4, entrapped proteins obtained under conditions favoring 8-1,4-glucan synthesis; F1.3, favoring P-1,3-glucan synthesis; F1,4, favoring P-1,4-glucan synthesis; Gx, an unknown compound made from GTP; 5’-N3, 5’-azido; PME, plasma membrane-enriched fraction; SE, solubilized enzyme.

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1150 Li et al. Plant Physiol. Vol. 101, 1993

MATERIALS A N D METHODS

Chemicals

[32P]Phosphate was purchased from ICN Biochemicals Inc. (Costa Mesa, CA). Cyclic diguanylic acid was offered by Dr. J.H. van Boom of the Gorlaeus Laboratories (Department of Organic Chemistry, Leiden, The Netherlands). UDP-Glc py- rophosphorylase, Suc phosphorylase, inorganic pyrophos- phorylase, benzyl DEAE-cellulose, nucleotides, protease inhibitors, and other chemicals were obtained from Sigma (St. Louis, MO). Trichoderma reesei CBH I was obtained from Novo Industri (A/S, Copenhagen/Denmark). Rhizopus arrhizus ,8-1,3-glucanase was a gift from Dr. E.T. Reese of U.S. Army Laboratories (Natick, MA). The molecular mass stand- ards, which contain phosphorylase b (97 kD), BSA (66 kD), ovalbumin (42 kD), carbonic anhydrase (31 kD), soybean trypsin inhibitor (21 kD), and hen egg white lysozyme (14 kD) were purchased from Bio-Rad (Richmond, CA).

Synthesis of [32P]5’-N3-UDP-Clc

5‘-N3-UTP was synthesized according to the method of Evans and Haley (1987). [,8-32P]5’-N3-UDP-Glc was synthesized by the procedure of Drake et al. (1989) with the following modifications: (a) [32-P]Glc-1-P was prepared by incubation of the following reaction mixture at room temperature for 10 min: 20 mM Tris-HC1 (pH &O), 25 mM SUC, 20 mM MgC12, 5 mM Na2HP04, 2 units of Suc phosphorylase, and 5 mCi of [32P]phosphate in a final volume of 75 pL; (b) purification of synthesized [,8-32P]5’-N3-UDP-Glc was performed by using a benzyl DEAE-cellulose column (10 x 1 cm) equilibrated with 0.01 M NH4HC03 (pH 8.6, prepared directly from NH4HC03, not adjusted with acid or base) (25 pL of 3% H202/100 mL). An ammonium bicarbonate gradient (260 mL of 0.01-0.35 M NH4HC03, 25 pL of 3% H202/100 mL) was used as the elution solution.

Plant Materials

The cotton line Gossypium hirsutum Texas marker 1 (TM- 1) was grown, bolls were harvested, and locules were re- moved and stored in liquid nitrogen under conditions previously described (Okuda et al., 1993).

Preparation, Solubilization, and Differential Entrapment of Clucan Synthases

The PME and the digitonin-solubilized enzyme fraction were prepared as previously described (Okuda et al., 1993). ,8-1,4-Glucan synthase was entrapped in a reaction mixture of F1,4, which contained 10 m bis-trispropane-Hepes (pH 7.6), 20 mM cellobiose, 8 mM MgC12, 1 mM CaC12, 100 p~ c- 3’:5’-GMP, 0.05% digitonin, 1.5 to 3.0 mg/mL of solubilized enzyme proteins, 4 units/mL of P-1,3-glucanase from Rhizo- pus arrhizus, and 1 mM UDP-Glc. ,8-1,3-Glucan synthase was entrapped in a reaction mixture of F1,3. This mixture contained 10 mM bis-trispropane-Hepes (pH 7.4), 20 mM cellobiose, 2 mM CaC12, 0.05% digitonin, 1.5 to 3.0 mg/mL of solubilized enzyme proteins, 4 units/mL of CBH I, and 1 m UDP-Glc.

The entrapment reactions were performed by incubation

at 25OC for 30 min and terminated by cooling the reaction mixtures on ice for 30 min. The reaction mixtures were centrifuged at 7000g for 40 min at 4OC. The pellets were resuspended in a small volume of buffer (50 mM Tris-HC1 [pH 7.41, 1 mM EDTA, 20% glycerol, and 0.1% digitonin) and homogenized with a Teflon homogenizer. The suspension was centrifuged at 7000g for 20 min. The resulting pellets were resuspended, rehomogenized, and recentrifuged twice. The final pellets were designated EPF1,., and EPFI,3. The protein concentration of each fraction was determined by a modification of the Lowry procedure (Markwell et al., 1978). Enzyme assays were performed as previously described (Li and Brown, 1993).

Photoaffinity Labeling

The mixture for the photolabeling reactions of F1,4 is the same as described earlier (Li et al., 1993; Okuda et al., 1993) except that [p-32P]5’-N3-UDP-G1~ was used as the substrate. Furthermore, this mixture is identical with that used for the entrapment described above, except for the absence of hy- drolase and the use of the azido probe. This mixture contained 10 mM bis-trispropane-Hepes (pH 7.6), 20 mM cellobiose, 8 mM MgC12, 1 mM CaC12, 100 PM c-3’:5’-GMP, 0.05% digitonin, the indicated quantity of membrane proteins, and 20 p~ [/3-32P]5’-N3-UDP-G1~ (specific activity, 2.5 mCi/pmol) in a final volume of 100 pL. The mixture for the photolabeling reactions of F1,3 was composed of 10 mM bis-trispropane- Hepes (pH 7.4), 20 mM cellobiose, 2 mM CaC12, 0.05% digitonin, the indicated quantity of enzyme proteins, and 20 p~ [p-32P]5’-N3-UDP-Glc. The reaction mixtures were preincu- bated in an open Eppendorf tube at room temperature for 1 min and then irradiated (short wavelength UV, 254 nm) with a hand-held UV lamp (Ultraviolet Products, San Gabriel, CA) from the top of the tube at a distance of 4 cm for 3 min on an ice surface. Reactions were stopped by addition‘of 0.4 mL of methanol, and protein pellets were collected using the methods of Wessel and Flügge (1984). The pellets were resuspended in an SDS sample buffer and heated in a boiling water bath for 10 min. Electrophoresis was performed as described by Porzio and Pearson (1977). Labeled polypeptides were detected by autoradiography at -8OOC using Ko- dak X-Omat AR film and a DuPont Cronex intensifying screen.

The quantitation of the photoaffinity insertion was camed out by greyvalue scanning of an autoradiogram of the gel. The autoradiogram was digitized and recorded using an Interactive Image Analysis System (IBAS) (Carl Zeiss, Ger- many). A low-pass filter was used, and the background of the image was subtracted. Then, a single-pixel densimetric trace was made diagonally across each photolabeled protein band, because some protein bands had variable widths. The area under each curve was calculated to obtain Kd and K, values.

RESULTS

Differential Entrapment and Photolabeling

Based on our studies of regulation and product analysis (Li and Brown, 1993; Okuda et al., 1993), we realized that, if we



(3-Clucan Synthases from Cotton 1151

altered the conditions in the reaction mixture in a specifiedway, different /3-glucan products would be synthesized. Wealso assumed that, if different products were synthesized bydifferent enzymes or the same enzyme, the enzyme(s) wouldbe captured by the differential entrapment and detected byaffinity photolabeling. The major variances in the differentialentrapments were (a) to entrap /3-1,3-glucan synthase in thepresence of CBH I (to specifically release cellulose and thecellulose-associated enzyme) and without magnesium and c-3':5'-GMP and (b) to entrap /3-1,4-glucan synthase withmagnesium and c-3':5'-GMP and in the presence of ,3-1,3-glucanase (to specifically release callose and the callose-associated enzyme).

Enzyme assays showed that the ANIP was enriched inEPFi,4 and /?-l,3-glucan was enriched in EPFi,3 (Table I). Theprotein profile from differential entrapment is shown inFigure 1, upper. A 52-kD polypeptide is the most abundantin both EPFu and EPF,,4.

Photolabeling of these proteins showed that a 37-kD poly-peptide was specifically labeled under the conditions of Fi,4(Fig. 1, lower, lane 6), although it was poorly detected withCoomassie blue (Fig. 1, upper, lane 6). In contrast, twopolypeptides at 52 and 66 kD were enhanced under theconditions of Fi,3 (Fig. 1, lower, lane 8). A polypeptide at 66kD in the PME and the SE was intensively labeled under thecondition without UV (Fig. 1, lower, lanes 1 and 3), indicatingan occurrence of nonspecific binding. This nonspecific UDP-Glc-binding polypeptide was ruled out by product entrap-ment under conditions of either Fi,3 or Fi.4 (Fig. 1, lower,lanes 5 and 7), giving evidence that this polypeptide is notspecifically associated with glucan synthesis. Note that an-other polypeptide at 66 kD was labeled in the entrapmentfraction under UV conditions and was enhanced by thecondition of Fi,3 (Fig. 1, lower, lane 8). This 66-kD polypep-tide is believed to be different from the nonspecific UDP-Glc-binding polypeptide.

Effect of Cations on Affinity Photolabeling

The optimal combination of effectors was not only impor-tant for incorporation of Glc into the glucan products (Li andBrown, 1993) but also was important for for UDP-Glc bind-ing. EPFi-4 is shown in Figure 2, lanes 1 to 4, and EPFj,3 ispresented in Figure 2, lanes 5 to 7. Lane 1 represents aphotolabeling reaction in the reaction mixture of Fi,4. Lane 2indicates that all of the photolabeled bands in lane 1 almosttotally disappear when magnesium and calcium are omitted

Table I. Glucan synthase activity in different enzyme fractions

KD

Fraction ANIP Ethanol-lnsolubleProduct

nmol ol Clu incorporated min~mg~' of protein

PMSEEPF,,3EPF,,4

1.30.8

n.d.a3.7

34.017.371.711.1

97-

66-

45-

31-

KD

SE EPF1 4 EPF1 3

1 2 3 4 5 6 7 8

PM SE EPF1 4 EPF1 3KD

!Bi*̂ x

I45-

31-

UV

Figure 1. Upper, Protein profiles of different enzyme fractionsdetected with Coomassie blue staining. Lanes 1 and 2, PME, 20 jig/lane; lanes 3 and 4, SE, 15 Mg/lane; lanes 5 and 6, EPF,,4, 5 Mg/lane;lanes 7 and 8, EPF,,3, 5 Mg/lane. Lower, Photolabeling of differentenzyme fractions. Lanes 1 and 2, PME; lanes 3 and 4, SE; lanes 5and 6, EPFM; lanes 7 and 8, EPFi,3. Lanes 1 to 6, Photolabeled inthe reaction mixtures of F,,4, lanes 7 and 8, photolabeled in thereaction mixtures of F, 3. Note that a 37-kD polypeptide was spe-cifically photolabeled in PME, SE, and EPF,,4, but not in EPFi,3,whereas the photolabeling of the polypeptides at 52 and 66 kDwas enhanced in EPF1f3.

" n.d., Not detected. www.plantphysiol.orgon April 13, 2019 - Published by Downloaded from

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I— EPF1-4 *H— EPF1-3 *-KD

97-1

66-1

45-

KD

-52

the conditions of Fi,4. Three bands (52, 66, and 32 kD) werefound to be calcium dependent. The relationship among thesethree bands is still unclear, but it is expected to be clarifiedby N-terminal amino acid sequencing.

Saturation Tests

The saturation tests for the UDP-Glc-binding componentsof cotton fiber glucan synthases are summarized in Figure 3.Greyvalue scanning of this figure showed that the 37-kDpolypeptide approached saturation with [/3-32P]5'-N3-UDP-Glc with an apparent Ka of 17 ^M. The 52-kD polypeptideapproached saturation with the probe with an apparent Kjof 18 HM. Other polypeptides (32, 66, and 93 kD) did not

-37

31-

1

+

2 3 4 5 6 7

+ + + + + +CBC-3':5'-GMP + + + + - - -Mg++ + - + - - - +Ca++ + - + +EDTA - + . . . . .EGTA - - + . - + +

Figure 2. Effect of cations on photoaffinity labeling. Lanes 1 to 4,EPFM; lanes 5 to 7, EPFU. Photolabeling conditions are indicatedbelow the figure. Note that magnesium is a critical factor for thephotolabeling of the 37-kD polypeptide. Conditions with calciumbut without magnesium are favorable to the photolabeling of the52-kD polypeptide.

and EDTA is added to the reaction mixture. The density ofthe 37-kD band was reduced when calcium was omitted fromthe reaction mixture (lane 3), indicating a positive rather thana negative regulatory function of this cation in the photo-labeling of the 37-kD polypeptide.

Magnesium was critically important for the differentialentrapment of the 37-kD polypeptide and its subsequentphotolabeling. When magnesium was absent in the reactionmixture, the 37-kD band was neither present nor photola-beled, whereas the 52- and 66-kD bands were enhanced.

Photolabeling in the reaction mixture of Fi,3 gave intenselyphotolabeled bands at 52 and 66 kD but nothing at the37-kD position (lane 5). When calcium was omitted fromthe reaction mixture, the 52- and 66-kD bands disappeared(lane 6). A faint band at 37 kD was observed in EPFi,swhen magnesium was added to the reaction mixture ofF,,3 (lane 7).

In Figure 2, two other bands at 93 and 32 kD wereobserved, but none of them was magnesium dependent. Likethe 52-kD polypeptide, the 32-kD band also was sensitive tocalcium (lane 6).

It is clear that the 37-kD polypeptide is magnesium de-pendent and it is enriched by differential entrapment under

31-

QPEWMJUE

KD

45-

-52

I—37

1 2 3 4 5 6

LANES

1Ch

3 4

Figure 3. Saturation with [32-P]5'-N3-UDP-Clc. Concentrations ofthe azido probe in photolabeling of proteins in EPFi,4: lane 1, 2 MM;lane 2, 5 MM; lane 3, 10 MM; lane 4, 20 MM; lane 5, 40 MM; lane 6, 60MM. A, Autoradiogram of photolabeling patterns; B, greyvalue scan-ning of the photolabeling of the 37-kD polypeptide; C, greyvaluescanning of the photolabeling of the 52-kD polypeptide.



/3-Glucan Synthases from Cotton 1153

reach saturation in the range of concentrations tested. Thissuggests that these three bands should have higher Kd valuesthan the 52- and 37-kD bands.

Competition Experiments

Competition experiments were carried out by using cold(unlabeled) UDP-Glc at concentrations ranging from 0 to 500MM in the presence of 20 MM [/J-32P]5'-N3-UDP-GIc (Fig. 4).The unlabeled UDP-Glc was competitive with the photoin-sertion of the labeled UDP-Glc probe into the 37-kD poly-peptide. For the 37-kD polypeptide, an apparent K( wasdetermined as 18 MM by greyvalue scanning. Unlike the 37-kD polypeptide, the 52-kD polypeptide was much less sen-sitive to unlabeled UDP-Glc. An apparent K{ of 96 MM wasdetermined for this band. Similar to the 52-kD polypeptide,the 32-kD polypeptide was also less sensitive to unlabeledUDP-Glc.

KD

31-A

1

GREYVALUE

,'"X ,,

\ / \LANES

GREYVALUE

LANESc

Figure 4. Competition with unlabeled UDP-Clc. Concentrations ofunlabeled UDP-Clc in photolabeling of proteins in EPFM: lane 1,no UDP-Clc; lane 2, 5 MM; lane 3, 10 MM; lane 4, 25 MM; lane 5, 50MM; lane 6, 100 MM; lane 7, 500 MM. A, Autoradiogram of photo-labeling patterns; B, greyvalue scanning of the photolabeling of the37-kD polypeptide; C, greyvalue scanning of the photolabeling ofthe 52-kD polypeptide.

KD

-437

B

1

*--«NMk «*** * 4

2 3 4 5 6 7 8

Figure 5. Effects of pH on the photolabeling of the 37- and 52-kDpolypeptides. A, Reaction mixtures of FM. Lane 1, pH 6.0; lane 2,pH 6.6; lane 3, pH 7.0; lane 4, pH 7.2; lane 5, pH 7.6; lane 6, pH8.0; lane 7, pH 8.4. B, Reaction mixtures of F,,3. Lane 1, pH 6.0;lane 2, pH 6.6; lane 3, pH 7.0; lane 4, pH 7.2; lane 5, pH 7.4; lane6, pH 7.6; lane 7, pH 8.0; lane 8, pH 8.4.

Effect of pH on Photoaffinity Labeling

The 37-kD polypeptide reached maximal photolabelingwithin a pH range of 7.6 to 8.0 (Fig. 5A), and the maximumphotolabeling of the 52-kD polypeptide was reached withina pH range of 7.4 to 7.6 (Fig. 5B). The effect of pH onphotolabeling parallels that of Glc incorporation (Li andBrown, 1993).

Putative Activators and Inhibitors

The effects of putative activators and inhibitors on photo-labeling are shown in Figure 6. Lane 1 represents the photo-labeling conditions without both cellobiose and c-3':5'-GMPin the reaction mixture. All photolabeled bands in this lanewere very faint. Lane 2 represents the photolabeling condi-

KD

97-

66-

45-

KD

-•93

-•52

-37-32

1 2 3 4 5 6

Figure 6. Tests for putative activators. Lane 1, No activators; lane2, with cellobiose but without c-3':5'-CMP; lane 3, with c-3':5'-CMP but without cellobiose; lane 4, with cyclic diguanylic acid butwithout cellobiose; lane 5, with crude Gx from A. xylinum; lane 6,with cellobiose and c-3':5'-GMP. Note massive labeling of the 93-kD polypeptide in lane 2. www.plantphysiol.orgon April 13, 2019 - Published by Downloaded from




tions with cellobiose but without c-3':5'-GMP in the reactionmixture. It was surprising to us that the photolabeling of a93-kD polypeptide was greatly enhanced under these con-ditions. Lane 3 represents the photolabeling conditions with-out cellobiose but with c-3':5'-GMP. Here, the photolabelingof the 93-kD polypeptide was considerably reduced. Lane 4portrays the conditions without cellobiose but with cyclicdiguanylic acid. Here, the photolabeling of the 37-kD bandwas reduced somewhat compared with that in lane 3, butthe photolabeling of the 93-kD band was completely absent.Lane 5 depicts the conditions with crude Gx from A. xylinumbut without cellobiose. The photolabeling pattern appearsunique, which may result from other component(s) in crudeGx, because with pure cyclic diguanylic acid (lane 4) theprominent band at approximately 47 kD is absent. Lane 6represents the conditions of Fi,4 with the presence of bothcellobiose and c-3':5'-GMP. Here, the photolabeling of the93-kD band was much less than that in lane 2. This indicatesthat c-3':5'-GMP can depress the photolabeling of the 93-kD polypeptide; however, we do not understand the mech-anisms of this reduced photolabeling.

Effect of Glc-1-P and Glc-6-P on Photolabeling

The solubilized enzyme fraction from cotton is shown inFigure 7. When Glc-6-P was added to the reaction mixture,it appears to have had no effect; yet, Glc-1-P enhanced thephotolabeling of the 37-kD band. We do not understand thisenhancement at present. On the other hand, Glc-1-P andGlc-6-P did reduce the photolabeling of the 66-kD polypep-tide (lanes 3 and 4). Considering that the photolabeling atthe 66-kD position is UV independent in the PME and SEbut is UV dependent in the entrapment fractions (Fig. 1), weinfer that two polypeptides may exist with very close molec-ular masses. One of these might be phosphoglucomutase,which could be diminished by Glc-1-P or Glc-6-P, and the

45- T5

31 -H

-37

-32

1

Figure 7. Effect of Clc-1-P and Clc-6-P. Lane 1, F,,4 reaction mix-ture; lane 2, with 50 MM unlabeled UDP-GIc; lane 3, with 50 MM

Clc-1-P; lane 4, with 50 MM Clc-6-P. Note that the photolabeling ofthe 66-kD polypeptide is diminished by Glc-1-P and Clc-6-P.

second polypeptide might be involved in /3-1,3-glucan syn-thesis, which is enhanced by the conditions of Fi,3. It wasreported earlier that phosphoglucomutase was labeled non-specifically by [/3-32P]5'-N3-UDP-Glc (Drake et al., 1989).

DISCUSSION

Our results show that several polypeptides in the productentrapment fraction can be photolabeled with [/3-32P]5'-N3-UDP-Glc; however, among these polypeptides, only a 37-kDpolypeptide could be identified as the catalytic subunit ofcellulose synthase. This polypeptide meets three general re-quirements for demonstrating the validity of specific proteinbinding in photolabeling studies (Lin et al., 1990): (a) aspecific protein in an enzyme preparation is photolabeled bythe photoprobe, (b) specific photoincorporation at the activesite is measured by the ability of the probe to saturate thebinding sites and by prevention of photolabeling with thenative substrate at appropriate concentrations, and (c) thephotolabeling is dependent on the presence of activating lightto exclude nonspecific labeling. Furthermore, only the 37-kDpolypeptide meets the criteria we have established specifi-cally for the catalytic subunit of cellulose synthase in thisstudy.

There are three polypeptides at 32, 52, and 66 kD thatshare some common properties in the photolabeling studies.These polypeptides are present in the EPFi,3 fraction differ-entially entrapped under conditions of Fi,3. They are photo-labeled under the conditions with calcium and without mag-nesium in the incubation mixture.

The 52-kD polypeptide is considered to be the most likelycandidate for the catalytic subunit of /3-1,3-glucan synthasefor the following reasons: (a) it is the most abundant proteinin the entrapment fraction (Fig. 1, upper), and (b) its photo-labeling is the most enhanced among these three bands (32,52, 66 kD) under the conditions with calcium but withoutmagnesium in the incubation mixture (Fig. 2, lanes 4 and 5).Of course, we cannot exclude the possibility that the 32- and66-kD polypeptides may also be involved in callose biosyn-thesis. It is important to note that the photolabeling of the52-kD polypeptide is depressed by magnesium, whereas thesynthesis of /3-1,3-glucan is not inhibited by magnesium (Liand Brown, 1993).

Before the present investigation, several different ap-proaches had been utilized for identification of callose syn-thase. A substrate analog, UDP-pyridoxal, was used as alabeling probe, and a 42-kD polypeptide from mung bean(Read and Delmer, 1987) and some polypeptides at 54 to 57kD from red beet (Mason et al., 1990) were labeled. With[/3-32P]5'-N3-UDP-Glc as a photoaffinity probe, Frost et al.(1990) concluded that the 57-kD polypeptide of red beet isthe most likely candidate for the catalytic subunit of callosesynthase based on its enrichment during product entrapment,its pH optimum for labeling, and its requirements for effec-tors. Dhugga and Ray (1991a, 1991b) reported that a 55-kDpolypeptide is associated with /3-1,3-glucan synthase activityfrom pea by means of either a combination of glycerolgradient centrifugation, product entrapment, and immuno-logical approaches or a combination of IEF and immunolog-ical techniques. A 31-kD polypeptide was reported to be the www.plantphysiol.orgon April 13, 2019 - Published by Downloaded from



P-Glucan Synthases from Cotton 1155

catalytic subunit of glucan synthase from Glycine max by immunological approaches (Fink et al., 1990) and from Lolium multiflorum by immunological precipitation and photolabeling with a pyrimidine analog of UDP-Glc (Meikle et al., 1991).

Recently, a 52-kD polypeptide from cotton fibers was identified as the catalytic subunit of callose synthase by a combination of glycerol gradient centrifugation and direct photolabeling with [32P]UDP-Glc (Delmer et al., 1991). Our results have confirmed Delmer’s identification of the 52-kD polypeptide as the catalytic subunit for P-1,3-glucan synthase even though different techniques were used. We are aware that the photolabeling of the 52-kD polypeptide is depressed by magnesium (compare lanes 1 and 4 in Fig. 2 and see Delmer et al., 1991), yet magnesium does not reduce the incorporation rate from Glc to @-1,3-glucan, even though it is not an absolute requirement for P-1,3-glucan synthesis (Delmer et al., 1977; Henry and Stone, 1982; Delmer et al., 1984; Hayashi et al., 1987; Li and Brown, 1993). At least two possibilities should be considered: (a) the 52-kD polypeptide may be a fragment from the original catalytic subunit; therefore, some properties of the catalytic subunit may have been modified; or (b) other components that are not depressed by magnesium may also be involved in the synthesis of /3-1,3-glucan.

One difference between our photolabeling and the photolabeling by Delmer et al. (1991) is that an 84-kD polypeptide is always present with the 52-kD polypeptide in their photolabeling, whereas the 84-kD polypeptide is absent in our photolabeling runs; however, the 93-, 66-, and 32-kD polypeptides always are present with the 52-kD polypeptide. Another difference between the two photolabeling proce- dures is that, whereas the digitonin-solubilized protein fraction was not labeled in their case (Delmer et al., 1991), severa1 bands were labeled in our digitonin-solubilized protein fraction. These differences may be largely a result of the different preparation techniques.

It is difficult to compare the results from different plant species, especially when different techniques have been used, but we believe that the 57-kD polypeptide from red beet and the 55-kD polypeptide from pea are probably analogous to the 52-kD polypeptide from cotton fibers. Therefore, a11 three polypeptides are good candidates to be the catalytic subunit in callose biosynthesis.

From our results, photoaffinity labeling of the 93-kD polypeptide still is confusing, but it is interesting because the photolabeling of this band is greatly enhanced by cellobiose under the conditions with magnesium and calcium but without c-3’:5’-GMP in the incubation mixture. This condition is not typical for the photolabeling of /3-1,4-glucan synthase, in which case the c-3’:5’-GMP is included. The conditions for photoaffinity labeling of the 93-kD polypeptide also are atypical for the photolabeling of P-1,S-glucan synthase, in which the magnesium is omitted. In the complete incubation mixture of F1,4, the photolabeling of the 93-kD polypeptide is depressed by the presence of c-3’:5’-GMP. This result suggests that both cellobiose and c-3’:5’-GMP are somehow involved in the regulation of the photolabeling of the 93-kD polypeptide, with cellobiose having positive regulatory function and c-3‘:5’-GMP having negative regulatory function.

The mechanism of how c-3’:5’-GMP diminishes the stim- ulation of cellobiose remains unclear. It is unlikely that c-3’:5’-GMP competes with cellobiose at the same binding site, because the molecular structures of these two com- pounds are so different. It may be reasonable to propose that c-3’:5’-GMP changes the conformation of the protein in some way so that the activation by cellobiose becomes inef- fective. When enzyme fractions are photolabeled in a typical incubation mixture of F1,3 in which everything is the same except the omission of magnesium, the labeling of the 93-kD polypeptide is not greatly enhanced. This indicates that stim- ulation of the photolabeling of the 93-kD polypeptide by cellobiose occurs only when magnesium is present. Further investigation is required to understand the function of the 93-kD polypeptide.

Recently, we obtained an N-terminal amino acid sequence from an unblocked region of the 52-kD polypeptide. These data will be useful in the construction of oligonucleo- tide probes leading toward the eventual identification of the gene(s) responsible for- callose biosynthesis in cotton. For cellulose synthase, the N-terminal amino acid sequencing of the 37-kD polypeptide is in progress and should provide critica1 data for the isolation of the gene for this polypeptide.

ACKNOWLEDCMENTS

We thank Richard Santos for assistance in printing the pictures and Jong Lee for assistance in using the Interactive lmage Analysis System. We thank Dr. Krystyna Kudlicka for helpful discussions and for editing the manuscript. We thank Dr. Martin Schulein of Novo Industri for the CBH I enzyme.

Received October 20, 1992; accepted January 20, 1993. Copyright Clearance Center: 0032-0889/93/101/1149/08.

LITERATURE ClTED

Amor Y, Mayer R, Benziman M, Delmer D (1991) Evidence for a cyclic diguanylic acid-dependent cellulose synthase in plants. Plant Cel l3 989-995

Delmer DP (1987) Cellulose biosynthesis. Annu Rev Plant .Physiol

Delmer DP, Heiniger U, Kulow C (1977) UDP-g1ucose:glucan syn- thetase in developing cotton fibers. I. Kinetic physiological properties. Plant Physiol 5 9 713-718

Delmer DP, Solomon M, Read SM (1991) Direct photolabeling with [32P]UDP-glucose for identification of a subunit of cotton fiber callose synthase. Plant Physiol95 556-563

Delmer DP, Stone BA (1988) Biosynthesis of plant cell walls. In J Priess, ed, The Biochemistry of Plants, Vol 14. Academic Press, Orlando, FL, pp 373-420

Delmer DP, Thelen M, Marsden MPF (1984) Regulatory mecha- nisms for the synthesis of @-glucans in plants. In WM Dugger, S Bartnicki-Garcia, eds, Structure, Function, and Biosynthesis of Plant Cell Walls. American Society of Plant Physiologists, Rock- ville, MD, pp 133-149

Dhugga KS, Ray PM (1991a) Isoelectric focusing of plant plasma membrane proteins. Further evidence that a 55 kilodalton polypeptide is associated with P-1,3-glucan synthase activity from pea. Plant Physiol95 1302-1305

Dhugga KS, Ray PM (1991b) A 55 kilodalton plasma membrane- associated polypeptide is involved in @-l,S-glucan synthase activity in pea tissue. FEBS Lett 278 283-286

Drake RR Jr, Evans RK, Wolf MJ, Haley BE (1989) Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites. J Biol Chem

3 8 259-290

264 11928-11933




Evans RK, Haley BE (1987) Synthesis and biological properties of 5-azido-Z’-deoxyuridine 5 ’-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. Biochemistry 26:

Fink J, Jeblick W, Kass H (1990) Partia1 purification and immunological characterization of 1,3-@-glucan synthase from suspension cell of Glycine mm. Planta 181: 343-348

Frost DJ, Read SM, Drake RR, Haley BE, Wasserman BP (1990) Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5- azido-UDP-glycose J Biol Chem 265: 2162-2167

Hayashi T, Read SM, Russell J, Thelen M, Lin FC, Brown RM Jr, Delmer DP (1987) UDP-glucose:(l-3)-P-glucan synthases from mung bean and cotton. Plant Physiol83: 1054-1062

Henry RJ, Stone BA (1982) Factors influencing P-glucan synthesis by particulate enzymes from suspension-cultured Lolium multiflorum endosperm cells. Plant Physiol 69: 632-636

Jacobs SR, Northcote D (1985) In vitro glucan synthesis by mem- branes of celery petioles: the role of the membrane in determining the linkage formed. J Cell Sci Suppl 2: 1-11

Kauss H (1987) Some aspects of calcium-dependent regulation in plant metabolism. Annu Rev Plant Physiol38: 47-72

Li L, Brown RM Jr (1993) 8-Glucan synthesis in the cotton fiber. 11. Regulation and kinetic properties of .&glucan synthases. Plant Physiol 101: 1143-1 148

Lin FC, Brown RM Jr, Drake RR Jr, Haley BE (1990) Identification of the uridine 5’-diphosphoglucose (UDP-GIc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J Biol Chem 2 6 5 4782-4784

Markwell MAK, Hass SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206-210

269-276

Mason TL, Read SM, Frost DJ, Wasserman BP (1990) Inhibition and labeling of red beet uridine 5’ diphosphoglucose:(l,3)-P-

. glucan (callose) synthase by chemical modification with formal- dehyde and uridine 5’ diphosphopyridoxal. Physiol Plant 79: 439-447

Mayer R, Ross P, Weinhouse H, Amikam D, Volman G, Ohana P, Calhoon RD, Wong HC, Emerick W, Benziman M (1991) Poly- peptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically cross- reacting proteins in higher plants. Proc Natl Acad Sci USA 88:

Meikle PJ, Ng KF, Johnson E, Hoogenraad NJ, Stone BA (1991) The P-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a nove1 photoreactive pyrimidine analog of uridine 5’-diphosphoglucose. J Biol Chem 266 22569-22579

Okuda K, Li L, Kudlicka K, Brown RM Jr (1993) P-Glucan synthesis in the cotton fiber. I. Identification of p-1,4- and P-1,3-glucans synthesized in vitro. Plant Physiol 101: 1131-1142

Porzio MA, Pearson AM (1977) Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacryamide gel electrophoresis. Biochim Biophys Acta 490: 27-34

Read SM, Delmer DP (1987) Inhibition of mung bean UDP-glucose:(l-3)-@-glucan synthase by UDP-pyridoxal. Plant Physiol85

Read SM, Delmer DP (1991) Biochemistry and regulation of cellulose synthesis in higher plants. In CH Haigler, PJ Weimer, eds, Biosynthesis and Biodegradation of Cellulose. Marcel Dekker, New York, pp 177-200

Wessel D, Fliigge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergent and lipids. Anal Biochem 138 141-143

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P-Glucan Synthesis in the Cotton Fiberâ€™