oxidative cross-linking of plasma membrane arabinogalactan proteins

The Plant Journal (1997) 12(5), 1189-1196

SHORT COMMUNICATION

Oxidative cross-linking of plasma membrane arabinogalactan proteins

Per Kjellbom 1,*, Lars Snogerup 1, Christine St6hr 1,t, Christophe Reuzeau 1, Paul F. McCabe 2,~, and Roger I. Pennell 2,§ 7Department of Plant Biochemistry, Lund University, PO Box 117, 221 O0 Lund, Sweden, and 2Department of Biology, University College London, Gower Street, London WCIE 6BT, UK

Summary

Monoclonal antibodies which recognize carbohydrate in arabinogalactan proteins (AGPs) have revealed that certain carbohydrate epitopes at the outer plasma membrane surface are developmentally regulated. Some epitopes are expressed according to cell position, and AGPs are thought to play a role in cell-cell interaction during development. This study demonstrates that sugar beet plasma membranes contain two subfamilies of AGPs, with apparent molecular masses of 82 and 97 kDa, and that each subfamily consists of a small number of acidic AGP isoforms. Excision of leaves generates three additional AGP complexes with apparent molecular masses of 120, 170 and 210 kDa, with the 170 kDa complex being the major form induced by excision. The addition of millimolar concentrations of H202 to a partially purified fraction of the 82 and 97 kDa AGPs also generates AGP complexes, with the 170 kDa complex as the major form. These results indicate that the plasma membrane AGPs are a target for endogenous H202.

Introduction

Many plant plasma membrane proteins are elaborately glycosylated and contribute to a complex carbohydrate- rich coating of the membrane. In mammalian cells, the carbohydrates of plasma membrane glycoproteins function as specific recognition structures for interaction at the

Received 4 March 1997; revised 12 June 1997; accepted 9 July 1997. tPresent address: Institut for Botanik, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, D-64287 Darmstadt, Germany. SPresent address: Plant Biology Laboratory, the Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186, USA. tPresent address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OXl 3RB, UK. Lars Snogerup and Christine St6hr contributed equally to this work. *For correspondence (fax +46 46 2224116; e-mail [email protected]).

outer surface of the membrane (Gahmberg and Tolvanen, 1996). This may also be true for glycoproteins of the plant plasma membrane (Brownlee and Berger, 1995) and extracellular matrix (Quatrano and Shaw, 1997).

One class of plant plasma membrane glycoproteins is the arabinogalactan proteins (AGPs). These are complex proteoglycans usually rich in alanine, serine, threonine and hydroxyproline residues in the core protein. The polysaccharide is composed of 13-1,3- and 1,6-1inked galactans containing uronides and neutral sugars including arabinose. AGPs are related to other hydroxyproline-rich glycoproteins (HRGPs) such as extensins and solanaceous lectins and they probably share common molecular ancestors (Fincher et aL, 1983; Kieliszewski and Lamport, 1994). However, AGPs differ in amino acid sequence motifs in the core protein, in carbohydrate content, composition and structure, and in the type of protein-carbohydrate linkages (Showalter, 1993). Several AGPs have been local- ized to the extracellular matrix (Roberts, 1994), although it is clear that some are true components of the plasma membrane (Komalavilas et aL, 1991; Norman et aL, 1990; Pennell etaL, 1989; Serpe and Nothnagel, 1996; Smallwood et aL, 1995; 1996; Tsumuraya et aL, 1990). In suspension cultures of rose cells, plasma membrane AGPs constitute about 6% of total plasma membrane protein and about 9% of the carbohydrate (Komalavilas et aL, 1991). Amino acid sequences deduced from genes coding for extracellular AGPs reveal secretion signals at the N-termini, and some also contain putative transmembrane domains, raising the possibility that extracellular AGPs could be released from the plasma membrane by proteolysis (Chen et aL, 1994; Duet aL, 1994; 1996; Mau et aL, 1995).

Monoclonal antibodies (mAbs) that recognize AGPs have revealed that some carbohydrate epitopes are developmentally regulated and mark cell position. In carrot roots, the epitope recognized by the mAb JIM4 occurs only on two cell collectives that are centred on the poles of the protoxylem and include future pericycle, xylem and endo- dermis cells (Knox et aL, 1989). In pea flowers, the epitope recognized by the mAb MAC207 specifies the somatic cells up until the time when the germ lines are established (Pennell and Roberts, 1990). In rapeseed, the epitope recognized by JIM8 specifies certain sexual cells and certain somatic cells that surround them (Pennell et aL, 1991). The spatial and temporal relationships between different AGP epitopes have led to the suggestion that AGPs are part of a cell-cell signalling mechanism that is involved with

1189

1190 Per K j e l l b o m et al.

cont ro l l ing cel lu lar d i f fe rent ia t ion (Knox, 1995; Kreuger and

van Hoist, 1996). It has been repor ted that ox ida t i ve cross-

l ink ing of pro l ine-r ich prote ins (PRPs) and HRGPs in the

cell wa l l leads to cell wa l l t oughen ing , and that this cross-

l inking is regula ted both in d e v e l o p m e n t and defence

(Bradley et aL, 1992; Brisson et aL, 1994). Our results

indicate that AGPs of the p lasma m e m b r a n e behave in a

s imi la r way.

A bet ter character izat ion of p lasma m e m b r a n e AGPs

shou ld help to exp la in AGP funct ion, and in turn this may

help to discern the mechan isms of cel l -cel l in teract ion in

p lant deve lopmen t . Here we repor t that sugar beet p lasma

membranes conta in t w o AGP subfami l ies, each consist ing

of a smal l n u m b e r of acidic isoforms, and that excis ion of

leaves causes these AGPs to cross-l ink and fo rm com-

plexes. We also demons t ra te that m i l l imo la r concent ra t ions

of H20 2 toge ther w i th perox idase induce AGP cross- l ink ing

in v i t ro when added to a par t ia l ly pur i f ied p lasma mem-

brane AGP fract ion.

Results

Sugar beet leaves were excised at 6°C and homogen ized

at 0°C, and p lasma membranes were pur i f ied by aqueous

p o l y m e r two-phase par t i t ion ing at 6°C. Tr i ton X-114 frac-

t i ona t ion of sugar beet p lasma membranes caused abou t

40% of the m e m b r a n e prote ins and all of the p lasma

m e m b r a n e AGPs to enter the wa te r f ract ion, as shown in

Figure l (a). The p lasma m e m b r a n e AGPs thus behave as

hydroph i l i c , wa te r -so lub le proteins.

Figure 1. Protein gel blots of sugar beet plasma membrane AGPs. (a) Immunoblots and Yariv reagent stain of Triton X-114 water (H) and detergent (X) fractions of plasma membranes resolved by SDS-AGE. The blots were probed with the mAbs MAC207 and JIM8 which recognize AGPs, and with the AGP-specific I~-glucosyl Yariv reagent. Aliquots of 10 ~g protein were loaded in the lanes probed with mAbs and 50 p.g aliquots in the lanes stained with Yariv reagent. (b) In vivo AGP cross-linking. Immunoblots, Yariv and Coomassie staining of the Triton X-114 water fraction of plasma membranes prepared from sugar beet leaves excised either at 6°C and homogenized at 0°C for plasma membrane purification and Triton X-114 fractionation (nw, non-wounded) or excised and kept at 22°C for 15 min prior to homogenization at 0°C for plasma membrane purification and Triton X-114 fractionation (w, excision- wounded). Proteins were either resolved by SDS-AGE and gel blots probed with MAC207 (left panel; 10 ~g protein in each lane), stained with Yariv reagent (left panel; 50 t~g protein) or resolved by SDS-PAGE and gel blots stained with Coomassie brilliant blue R-250 (right panel; 50 ~tg protein in each lane). (c) In vitro AGP cross-linking. H202- and peroxidase-dependent in vitro cross-linking of sugar beet plasma membrane AGPs. Aliquots of the Triton X-114 water fraction (5 ~tg protein) were incubated with horseradish peroxidase (0.1 mg m1-1) and different concentrations of H202, sodium azide (10 mM; an inhibitor of peroxidases), ascorbate (at concentrations equimolar to H202; a radical scavenger), and catalase (0.5 mg m1-1) for 60 min at 25°C. The samples were resolved by SDS-AGE and the blot was probed with MAC207. The numbers refer to apparent molecular mass of the plasma membrane AGPs (arrows) in kDa as compared with standard proteins (to the right in (a) and (b); high-molecular-weight standard proteins, Bio-Rad, Richmond, CA, USA).

Oxidative cross-linking of AGPs 1191

Aliquots of the water and detergent fractions obtained by Triton X-114 fractionation were resolved by sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) according to St6hr et aL (1996). Protein blots probed with the AGP-specific ligand 13-glucosyl Yariv reagent and two mAbs that recognize AGPs (MAC207 and JIM8) revealed two plasma membrane AGP bands at approximately 82 and 97 kDa, as shown in Figure l(a). The relatively weak Yariv staining is consistent with the staining of other AGPs from both the plasma membrane (Komalavilas et aL, 1991) and the cell wall (Serpe and Nothnagel, 1995; Smallwood et aL, 1996).

Excision of leaves at 22°C prior to homogenization and plasma membrane preparation resulted in five AGP bands as shown in Figure l(b). This suggested that the formation of the additional AGP bands of approximately 120, 170, and 210 kDa was a result of excision. The major AGP was the 170 kDa form (Figure lb). The formation of the 170 kDa AGP band could be mimicked in vitro by adding millimolar concentrations of H202 and horseradish peroxidase to a Triton X-114 water fraction containing only the 82 and 97 kDa plasma membrane AGPs (Figure lc). As revealed by MAC207 (Figure lb and c) and Yariv staining (Figure lb; data not shown), the 170 kDa AGP bands formed in vivo and in vitro had the same carbohydrate epitopes. Taken together, this suggested that the higher molecular mass forms appear as a result of cross-linking, with only the 170 kDa AGP band being formed in vitro. Optimum cross- linking was achieved with 1 mM H202, which is in agree- ment with the levels of H202 that are thought to accumulate during elicitor-induced oxidative burst by plant cells (Legendre et aL, 1993; Mehdy, 1994). H202 at 10 mM inhibited cross-linking relative to 1 mM, and 100 mM, which is a non-physiological concentration, abolished mAb binding (data not shown), probably by disrupting the epitopes recognized by the mAbs. Upon formation of the 170 kDa band in vitro, there was a decrease in the 82 kDa AGP band (Figure lc). The in vitro cross-linking was dependent on the presence of peroxidase and H202 (data not shown) and was inhibited by ascorbate, which is an oxygen radical scavenger, by sodium azide, which is an inhibitor of haem enzymes and therefore blocks peroxidase activity, and by catalase, which catalyses the breakdown of H202 to water and 02 (Figure lc). The relatively high catalase concentration is a requirement of the high K m of the enzyme and is similar to concentrations used in earlier studies (Behl et aL, 1994; Bradley et aL, 1992; Levine et aL, 1994; Wu et al., 1995). No major changes in the overall plasma membrane polypeptide pattern of the Triton X-114 fraction could be detected by SDS-PAGE following excision (Figure lb) or in vitrocross-linking using BSAas an internal control (data not shown). This suggests that, apart from the plasma membrane AGP cross-linking, no general cross-

Figure 2. Sugar beet AGP immunoblots and protoplast immunofluorescence. (a) Epitope distribution. Gel blots of Triton X-114 water (H) and detergent (X) fractions, prepared from plasma membranes of wounded leaves, resolved by SDS-AGE. The blots were probed with different mAbs that recognize AGPs. The numbers refer to apparent molecular mass of the plasma membrane AGPs in kDa compared with standard proteins (high- molecular-weight) standard proteins, Bio-Rad, Richmond, CA, USA). Aliquots of 10 p.g protein were loaded in each lane. (b) Sugar beet leaf protoplast immunofluorescence. Green fluorescence is due to the binding of the mAbs and red is due to chlorophyll autofluorescence.

linking of plasma membrane proteins occurs either in vivo or in vitro.

Figure 2(a) shows immunoblots of Triton X-114-fraction- ated detergent and water phases of plasma membranes, prepared from leaves excised and kept at 22°C prior to homogenization at 0°C, and probed with a panel of mAbs that recognize AGPs. The epitopes recognized by MAC207, JIM8, PN16.4B4 and Ox-FB8 are all present on the 82 and the 97 kDa AGPs, but the epitopes have different distributions on the AGP complexes. For example, only the 170 kDa complex could be labelled with JIM8, whereas all three AGP complexes were labelled with MAC207 and PN16.4B4 (Figure 2a). Ox-FB8 recognizes the 120 and 170 kDa complexes but not the 210 kDa complex. In contrast, the epitopes recognized by JIM14 and JIM15 are only found on the AGP complexes (Figure 2a). This is interesting because JIM14 and JIM15 both recognize developmentally regulated epitopes (Knox et aL, 1989; 1991). Immunofluorescence of protoplasts confirmed the presence of the epitopes at the plasma membrane, as seen for MAC207, JIM8 and JIM15 in Figure 2(b).

Since MAC207 and JIM8 reacted similarly with the 82 and the 97 kDa AGPs but differently with the 120 kDa AGP complex, we examined the plasma membrane AGPs for

1192 Per K j e l l b o m et al.

brane AGP epitopes does not vary from organ to organ. However, variability in the distribution of AGP isoforms may occur, as well as cell-specific differences in AGP epitope distribution within organs.

Figure 3. AGP isoforms differing in isoelectric point. MAC207 and JIM8 two-dimensional immunoblots. An aliquot of the Triton X-114 water fraction (20 pg protein), prepared from plasma membranes of non-wounded leaves, was resolved by isoelectric focusing in an immobilized pH gradient in the first dimension and SDS-AGE in the second dimension. The 82 kDa AGP isoform carrying both the MAC207 and the JIM8 epitope is marked with an arrowhead; the other four 82 and 97 kDa isoforms are marked with arrows. The numbers on the vertical axis refer to the apparent molecular mass of the plasma membrane AGPs in kDa compared with standard proteins (high-molecular-weight standard proteins, Bio-Rad, Richmond, CA, USA). The numbers on the horizontal axis denote pH and the arrow indicates the sample application point in the first dimension.

evidence of AGP isoforms. As seen in Figure 3, two- dimensional immunoblots revealed that four acidic isoforms of the 82 and the 97 kDa AGPs were labelled with MAC207 and two with JIM8. One of the 82 kDa isoforms (indicated by arrowheads in Figure 3) was labelled with both mAbs, suggesting a total of five isoforms for the 82 and the 97 kDa AGPs as detected by MAC207 and JIM8. The 170 kDa complex also appeared as several discrete forms, although the resolution was less good because the AGP complexes tend to precipitate at the point of application in the isoelectric focusing gel (StOhr et al., 1996). It is therefore not clear whether the 170 kDa isoforms have migrated to their true pl (Figure 3). Like the sample applied to the SDS-AGE gel in Figure l(a), the sample applied to the two-dimensional gels in Figure 3 was from plasma membranes of non-wounded leaves and contained the 82 and the 97 kDa AGPs and traces of the 170 kDa AGP complex. This suggests that plasma membranes in non- wounded leaves contain two major subfamilies of plasma membrane AGPs and that each subfamily consists of a small number of acidic isoforms.

As analysed by SDS-AGE, the same set of MAC207 reactive bands present in leaf plasma membranes (Figure la, b) was also present in the plasma membranes of all other organs tested (flower, sepal, petiole, root; data not shown). This was also true for rapeseed organs (data not shown), suggesting that the presence of plasma mem-

Discussion

As far as we know, the Yariv reagent and the mAbs used in this study together detect all the AGPs that are present in sugar beet plasma membranes (Pennell et al., t991). Immunoblots of Triton X-114 detergent and water fractions show that plasma membrane AGPs are hydrophilic in nature, probably because of the large amount of negatively charged polysaccharide. Based on reverse-phase chroma- tography elution profiles, Serpe and Nothnagel (1996) recently reported that crude plasma membrane AGP fractions of rose suspension culture cells are not more hydrophobic than cell wall AGPs. However, these results do not necessarily mean that plasma membrane AGPs do not contain transmembrane domains, since the polysaccharide could probably shield such domains from thermo- dynamically unfavourable interactions with water (Bricker and Sherman, 1982). The observation that only a small amount of AGP is released from right-side-out or inside- out plasma membrane vesicles by high salt, or high or low pH washes (Norman et al., 1990; data not shown) is consistent with the view that plasma membrane AGPs are anchored by non-ionic bonds. In addition to N-terminal secretion signals, the deduced amino acid sequences of some of the genes encoding extracellular AGPs have putative transmembrane domains at their C-termini (Chen et al., 1994; Duet al., 1994; Li and Showalter, 1996). It is not clear whether these transmembrane domains are still present in extracellular AGPs, and it has not been demonstrated that extracellular AGPs are derived from AGPs at the plasma membrane. In contrast to our results, which show that sugar beet plasma membrane AGPs are hydrophilic, the AGPs in rice are hydrophobic (Smallwood etal., 1996). This could be due to more extensive stretches of hydrophobic amino acids, or incompletely glycosylated AGPs originat- ing from intracellular membranes such as the endoplasmic reticulum.

Our results suggest that leaf excision causes plasma membrane AGPs of the two AGP subfamilies to cross-link and form high-molecular-weight complexes. While de novo synthesis induced by excision could account for the high- molecular-weight AGPs, the short incubation time at 22°C following excision suggests that this is less likely than cross-linking. According to the MAC207 immunoblot and the Yariv stain, the 170 kDa complex is the major AGP form induced by excision. The MAC207 blot shows that the other two AGP complexes (120 and 210 kDa bands) are less abundant. The 210 kDa AGP complex was consistently found to stain relatively weakly with the Yariv reagent. This

is probably due to the lower sensitivity of this staining method together with the lower abundance of this complex. Alternatively, the 210 kDa complex could have relatively few binding sites for the Yariv reagent, as indicated for other plasma membrane (Komalavilas et al., 1991) and cell wall (Serpe and Nothnagel, 1995; Smallwood eta/., 1996) AGPs. In addition to AGP cross-linking following wounding by excision, there seems to be developmental control over AGP cross-linking. Preliminary data indicate that older leaves have more of the cross-linked AGP complexes than younger leaves (data not shown). This pattern is similar to that found during the oxidative cross-linking of a PRP and an HRGP in cell walls of bean (Bradley eta/., 1992). Apart from developmentally regulated cross-linking, these two proteins are cross-linked in the cell wall within 2 min following wounding.

The banding patterns of the AGP complexes were the same when reducing agents, e.g. mercaptoethanol and dithiothreitol, were present in the sample buffer (data not shown), excluding the possibility that disulphide bridges form AGP cross-links. The mechanisms of H202-dependent cross-linking of glycoproteins are not known, although intermolecular linkages involving tyrosine (Fry, 1986; Varner and Lin, 1989; Waffenschmidt eta/., 1993), or tyrosine and lysine (Schnabelrauch eta/ . , 1996), could be formed.

The cross-linking observed when a partially purified plasma membrane AGP fraction, containing the 82 and 97 kDa AGPs, is exposed to H202 and horseradish peroxidase shows that AGP cross-linking can be accomplished in vitro. Hydrogen peroxide substitutes for an active endogenous source of H202, such as the plasma membrane NAD(P)H oxidase (Askerlund eta/., 1987; Groom eta/., 1996) possibly in combination with an apoplastic superoxide dismutase, and horseradish peroxidase substitutes for an endogenous cell wall peroxidase (Schnabelrauch eta/., 1996). As for excision-induced cross-linking, it is the 170 kDa AGP complex that is predominant in vitro. The 120 and 210 kDa complexes that form in vivo are not formed in vitro, and may require additional proteins for cross-linking which are not present in the Triton X-114 water fraction used for in vitro experiments. The amount of 82 kDa AGP diminishes during H202-dependent complex formation in vitro, suggesting that H202 causes two 82 kDa AGP molecules to cross-link and form dimers that run at 170 kDa on SDS- AGE gels. However, it is also possible that 82 kDa AGPs cross-link to other plasma membrane proteins present in the Triton X-114 fraction to form the 170 kDa species. This possibility is also suggested by the two-dimensional immunoblot probed with MAC207 provided that the 170 kDa AGP species have migrated to their true pls; dimers of the 82 kDa AGP species should have pls around 4 rather than 6 as for the 170 kDa species. Also, dimers of the 97 kDa AGP species, and heterodimers of the 82 and

Oxidat ive cross-f inking o f AGPs 1193

97 kDa species, should have pls around 4. Since both the 82 and the 97 kDa AGPs occur as a small number of isoforms, it is also possible that only certain isoforms take part in the complex formation. One way to address the question of whether the 170 kDa AGP band represents homodimers of the 82 kDa AGPs would be to perform in vitro cross-linking with purified individual AGPs. Since we have been able to resolve individual plasma membrane AGPs (StShr et a/., 1996), it should be possible to generate the quantities necessary for testing self-cross-linking. Experiments are in progress to purify the AGP complexes and generate partial amino acid sequences of the proteins of which they are composed, in order to identify the proteins that cross-link to form these complexes.

Some AGP epitopes, such as the JIM14-reactive epitope, only occur on the AGP complexes. This suggests that H202 generation can guide the appearance of specific epitopes, and raises the possibility that there are two ways in which AGP epitopes can be controlled. These could involve glycosylation in the secretory pathway and normal membrane flow and membrane turnover, or accumulation of H202 and subsequent cross-linking of AGPs at the outer surface of the plasma membrane.

The AGP complexes that appear in vivo are likely to be dependent on H202 generated from superoxide during an oxidative burst (Lamb, 1994). In plant defence, H202 accumulates during the hypersensitive response (HR) to attempted invasion by incompatible plant pathogens, and is thought to play a central role in the defence mechanism (Levine eta/., 1994; 1996; Mehdy eta/., 1996; Wu eta/., 1995). One effect of the H202 accumulation is cross-linking of cell wall proteins, such as PRPs and HRGPs (Bradley et al., 1992; Brisson eta/., 1994; Smallwood et a/., 1995). This cell wall protein cross-linking leads to a strengthened cell wall and is thought to be a rapid defence response that operates prior to transcription-dependent defences (Brisson eta/., 1994). Our results suggest that H202 also causes cross-linking of AGPs.

We have shown that sugar beet plasma membranes contain two subfamilies of AGPs, each consisting of a number of acidic AGP isoforms, and that leaf excision, and H202 in combination with peroxidase, can cause AGP cross- linking. Apart from the putative role of AGPs in cell-cell interactions during development (Kreuger and van Hoist, 1996), the data presented here suggest that AGP cross- linking is involved in responses to wounding.

Experimental procedures

Plants and molecular probes

The AGP artificial antigen ~-glucosyl Yariv reagent (1,3,5-Tris [4-~-o-glucopyranosyl-oxyphenylazo]-2,4,6-trihydroxybenzene; Nothnagel and Lyon, 1986; Yariv et aL, 1962) was from Biosupplies

1194 Per K je l l bom et al.

Australia Ltd (Parkville, Australia). The anti-AGP mAbs were from immunizations with different kinds of plant cell preparations and extracts (Bradley et aL, 1988; Dewey et aL, 1997; Knox et al., 1989; Norman etaL, 1986; Pennell etal., 1989). Sugar beet (Beta vulgaris) cv. Hilma was grown from seed in natural light.

Plasma membrane preparation

Leaves were excised at 6°C and immediately homogenized at 0°C, or excised and kept at 22°C for 15 min prior to homogenization at 0°C. Plasma membranes were purified bytwo-phase partitioning (at 6°C; Kjellbom and Larsson, 1984; Larsson et al., 1987) of a microsomal membrane fraction (10 000 9-50 000 g pellet) with a mixture of Dextran T 500 (average Mr 500 000) (Pharmacia, Uppsala, Sweden) and polyethylene glycol 3350 (average M r 3350; Union Carbide, New York, NY, USA). The hydrophobic plasma membrane proteins were then separated from hydrophilic ones by fractionation in 1% (w/v) Triton X-114, 150 mM NaCI and 10 mM Tris-HCI, pH 7.5, and 1 mM EDTA (Bordier, 1981; Kjellbom et al., 1989). The hydrophobic proteins partition into the detergent phase and the hydrophilic proteins into the water phase. A mixture of protease inhibitors (0.1-1.0 mM final concentrations of pepstatin A, leupeptin, aminobenzamidine, phenylmethylsulfonyl fluoride, NotosyI-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone) was added to avoid proteolysis.

Immunoblot t ing

SDS-PAGE was performed according to Laemmli (1970). SDS- AGE was performed according to StOhr et al. (1996) using a resolving gel made from 5% MetaPhor agarose (FMC BioProducts, Rockland, ME, USA), and a stacking gel consisting of 1.5% SeaKem Gold agarose (FMC), with ExcelGel Buffer Strips (Pharmacia). Proteins (5-20 pg protein for immunoblots and 50 IJg for Coomas- sie and Yariv staining of gel blots) were solubilized in 62.5 mM Tris-HCI, pH 6.8, 2% SDS and 0.001% bromophenol blue. For Coomassie-stained SDS-PAGE gels, 5% ~-mercaptoethanol was added in the sample buffer (Laemmli, 1970). Two-dimensional electrophoresis was performed by isoelectric focusing in the first dimension using Immobiline DryStrips (Pharmacia), pH 3-10.5, with protein (20 I~g) solubilized in 8 M urea (United States Bio- chemical Corporation, Cleveland, OH, USA) containing 1% Nonidet P-40 (Sigma Chemical Company, Saint Louis, MO, USA) and 0.001% bromophenol blue. Before the second dimension, SDS- AGE, the strips were equilibrated in sample buffer (Laemmli, 1970). Western blotting was performed as described by Towbin et al. (1979). Polyvinylidene difluoride (PVDF) membranes (Micron Separations Inc., Westborough, MA, USA) were used and no methanol was added to the transfer buffer. Blots were blocked in 2% low-fat milk in TBS (10 mM Tris-HCI, pH 7.2, 150 mM NaCI) for 30 min and incubated overnight at 6°C with 1% solutions of hybridoma supernatants in the blocking solution. Bound antibody was detected with an alkaline phosphatase-conjugated anti-rat IgG (H+L) antiserum. The apparent molecular masss of the AGPs were determined compared with standard proteins (high-molecular-weight standard proteins, Bio-Rad, Richmond, CA, USA).

In vitro AGP cross-linking

Aliquots (5 ~g protein) of water fractions from Triton X-114 fractionations were incubated in 100 mM potassium phosphate, pH 6.0, in the presence of 0-100 mM H202 and 0.1 mg m1-1 horseradish peroxidase (Sigma), with or without the addition of

ascorbate at concentrations equimolar to H202, 10 mM sodium azide (inhibits haem enzymes), and 0.5 mg m1-1 catalase for 60 min at 25°C. The presence of horseradish peroxidase (and H202) was required for cross-linking to occur (data not shown). The relatively high catalase concentration is consistent with that used in previous studies (Behl et aL, 1994; Levine et al., 1994; Wu et al., 1995) and reflects the high K m of the enzyme. The water fractions were solubilized for 15 min at 22°C in sample buffer (Laemmli, 1970) and resolved by SDS-AGE.

Protoplast immunofluorescence

Protoplasts were isolated from leaves of 4-week-old plants. The midribs were removed and the laminae sliced into a modified W5 solution (Menczel et al., 1981), in which the glucose was replaced by an additional 5 mM KCI and the pH was adjusted to 7.0, containing 9% (w/v) mannitol and the enzymes cellulase (Onozuka R10) and macerozyme (Yakult Honsha Co., Tokyo, Japan). The digestions were performed at 25°C for 18 h. Protoplasts were sieved from undigested material through 125 and 60 F,m nylon meshes and washed in the W5 solution. The protoplasts were transferred to 0.63 M sucrose and overlaid with the W5 solution. Centrifugation at 100 g for 10 min caused the viable protoplasts to float into the W5 solution. Protoplasts were collected with a pipette. Both slicing of leaves and protoplast preparation induce wound responses in plants. For immunofluorescence, the protoplasts were resuspended in the modified W5 solution containing 0.5% (w/v) dried milk and 2% (v/v) of a mAb hybridoma culture supernatant and incubated for 2 h. After washing in the W5 solution, the protoplasts were incubated in 1% (v/v) of an FITC- conjugated goat anti-rat IgG (H+L) antiserum for 1 h. After washing, the protoplasts were photographed with a Zeiss Photo- microscope 3 (Carl Zeiss, Oberkochen, Germany) using epi- fluorescence.

Acknowledgements

We thank Adine Karlsson for skilful technical assistance, Keith Roberts, Michael Hahn, and Quentin Cronk for mAbs, and Hillesh6g/Sandoz Seeds for plants. The research was funded by the Swedish Council for Forestry and Agricultural Research, the Swedish Natural Science Research Council, the European Com- munities HCM Network Programme, the Carl Trygger Foundation for Scientific Research, and the Royal Society of London (R.I.P.). R.I.P. is a Royal Society University Research Fellow.

References

Askerlund, P., Larsson, C., Widell, S. and Meller, I.M. (1987) NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences. Physiol. Plant. 71, 9-19.

Behl, C., Davis, J.B., Lesley, R. and Schubert, D. (1994) Hydrogen peroxide mediates amyloid [3 protein toxicity. Cell, 77, 817-827.

Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604-1607.

Bradley, D.J., Wood, E.A., Larkins, A.P., Galfr(~, G., Butcher, G.W. and Brewin, N.J. (1988) Isolation of monoclonal antibodies reacting with peribacteroid membranes and other components of pea root nodules containing Rhizobium leguminosarum. Planta, 173, 149-160.

Bradley, D.J., Kjellbom, P. and Lamb, C.J. (1992) Elicitor- and wound-induced oxidative cross-linking of a plant cell wall proline-rich protein: a novel, rapid defense response. Ceil, 70, 21-30.

Bricker, T.M. and Sherman, L.A. (1982) Triton X-114 phase- fractionation of maize thylakoid membranes in investigation of thylakoid protein topology. FEBS Lett. 149, 197-202.

Brisson, L.F., Tenhaken, R. and Lamb, C.J. (1994) Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance. Plant Ceil, 6, 1703-1712.

Brownlee, C. and Berger, F. (1995) Extracellular matrix and pattern in plant embryos: on the lookout for developmental information. Trends Genet. 11,344-348.

Chen, C.-G., Pu, Z.-Y., Moritz, R.L., Simpson, R.J., Bacic, A., Clarke, A.Eo and Mau, S.-L. (1994) Molecular cloning of a gene encoding an arabinogalactan-protein from pear (t~rus comrnunis) cell suspension culture. Proc. NatlAcad. Sci. USA, 91, 10 305-10 309.

Dewey, EM., Thurston, M.I. and Cronk, Q.C.B. (1997) Monoclonal antibodies that differentiate between gum arabic, gum seyal and combretum gum. FoodAgric,/mrnuno/. 9, 123-124.

Du, H., Simpson, R.J., Moritz, R.L., Clarke, A.E. and Bacic, A. (1994) Isolation of the protein backbone of an arabinogalactan- protein from styles of Nicotiana a/ata and characterization of a corresponding cDNA. Plant Cell, 6, 1643-1653.

Du, H., Simpson, R.J., Clarke, A.E. and Bacic, A. (1996) Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein (AGP) from Nicotiana alata. Plant J. 9, 313-323.

Fincher, G.B., Stone, B.A. and Clarke, A.E. (1983) Arabinogalactan proteins: structure, biosynthesis and function. Annu. Rev. Plant Physiol. 34, 47-70.

Fry, S.C. (1986) Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annu. Rev. Plant Physiol. 37, 165-186.

Gahmberg, C.G. and Tolvanen, M. (1996) Why mammalian cell surface proteins are glycoproteins. Trends Biol. Sci. 21,308-311.

Groom, Q.J., Torres, M.A., Fordham-Skelton, A.P., Hammond- Kosack, K.E., Robinson, N.J. and Jones, J.D.G. (1996) rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene. Plant J. 10, 515-522.

Kieliszewski, M.J. and Lamport, D.T.A. (1994) Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny. P/antJ. 5, 157-172.

Kjellbom, P. and Larsson, C. (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant. 62, 501-509.

Kjellbom, P., Larsson, C., Rochester, C.P. and Andersson, B. (1989) Integral and peripheral proteins of the spinach leaf plasma membrane. Plant Physiol. Biochem. 27, 169-174.

Knox, J.R (1995) Developmentally regulated proteoglycans and glycoproteins of the plant cell surface. FASEB J. 9,1004-1012.

Knox, J.P., Day, S. and Roberts, K. (1989) A set of cell surface glycoproteins forms an early marker of cell position, but not cell fate, in the root apical meristem of Daucus carota L. Development, 106, 47-56.

Knox, J.P., Linstead, P.J., Peart, J., Cooper, C. and Roberts, K. (1991) Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation. Plant J. 1,317-326.

Komalavilas, R, Zhu, J.-K. and Nothnagel, E.A. (1991) Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells. J. Biol. Chem. 266, 15 955-15 965.

Kreuger, M. and van Hoist, G.-J. (1996) Arabinogalactan proteins and plant differentiation. Plant Mol. Biol. 30, 1077-1086.

Oxida t i ve c ross- l ink ing o f A G P s 1195

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.

Lamb, C.J. (1994) Plant disease resistance genes in signal perception and transduction. Cell, 76, 419-422.

Larsson, C., Widell, S. and Kjellbom, R (1987) Preparation of high- purity plasma membranes. Methods Enzyrnol. 148, 558--568.

Legendre, L., Reuter, S., Heinstein, RF. and Low, P.S. (1993) Characterization of the oligogalacturonide-induced oxidative burst in cultured soybean (G/ycine max) cells. Plant Physiol. 102, 233-240.

Levine, A., Tenhaken, R., Dixon, R. and Lamb, C. (1994) H202 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell, 79, 583-593.

Levine, A., Pennell, R.I., Alvarez, M.E., Palmer, R. and Lamb, C. (1996) Calcium-mediated apoptosis in a plant hypersensitive desease resistance response. Curt. Biol. 6, 427-437.

Li, S.-x. and Showalter, A.M. (1996) Cloning and developmental/ stress-regulated expression of a gene encoding a tomato arabinogalactan protein. Plant Mol. Biol. 32, 641-652.

Mau, S.-L., Chen, C.-G., Pu, Z.-Y., Moritz, R.L., Simpson, R.J., Bacic, A. and Clarke, A.E. (1995) Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of I~rus cornrnunis and Nicotiana alata. Plant J. 8, 269-281.

Mehdy, M.C. (1994) Active oxygen species in plant defense against pathogens. Plant Physiol. 105, 467-472.

Mehdy, M.C., Sharma, Y.K., Sathasivan, K. and Bays, N.W. (1996) The role of activated oxygen species in plant desease resistance. Physiol. Plant. 98, 365-374.

Menczel, L., Nagy, F., Kiss, Z.R. and Maliga, R (1981) Streptomycin- resistant and sensitive somatic hybrids of Nicotiana tabacurn + Nicotiana knightiana: correlation of resistance to N. tabacurn plastids. Theor. Appl. Genet. 59, 191-195.

Norman, P.M., Kjellbom, P., Bradley, D.J., Hahn, M.G. and Lamb, C.J. (1990) Imrnunoaffinity purification and biochemical characterization of plasma membrane arabino-galactan-rich glycoproteins of Nicotiana glutinosa. Planta, 181,365-373.

Norman, P.M., Wingate, V.P.M., Fitter, M.S. and Lamb, C.J. (1986) Monoclonal antibodies to plant plasma membrane antigens. Planta, 167, 452-459.

Nothnagel, E.A. and Lyon, J.L. (1986) Structural requirements for the binding of phenylglycosides to the surface of protoplasts. Plant Physiol. 80, 91-98.

Pennell, R.I. and Roberts, K. (1990) Sexual development in the pea is presaged by altered expression of arabinogalactan protein. Nature, 344, 547-549.

Pennell, R.I., Knox, J.P., Scofield, G.N., Selvendran, R.R. and Roberts, K. (1989) A family of abundant plasma membrane- associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants. J. Cell Biol. 108, 1967-1977.

Pennell, R.I., Janniche, L., Kjellbom, P., Scofield, G.N., Peart, J.M. and Roberts, K. (1991) Developmental regulation of a plasma membrane arabinogalactan protein in oilseed rape flowers. Plant Cell, 3, 1317-1326.

Quatrano, R.S. and Shaw, SoL. (1997) Role of the cell wall in the determination of cell polarity and the plane of cell division in Fucus embryos. Trends Plant Sci. 2, 15-21.

Roberts, K. (1994) The plant extracellular matrix: in a new expansive mood. Curr. Opin. Cell Biol. 6, 688-694.

Schnabelrauch, L.S., Kieliszewski, M., Upman, B.L., Alizedeh, H. and Lamport, D.T.A. (1996) Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val- Tyr-Lys as putative intermolecular cross-link site. Plant J. 9, 477-489.

1196 Per K je l l bom et al.

Serpe, M.D. and Nothnagel, E.A. (1995) Fractionation and structural characterization of arabinogalactan-proteins from the cell wall of rose cells. Plant Physiol. 109, 1007-1015.

Serpe, M.D. and Nothnegel, E.A. (1996) Heterogeneity of arabinogalactan-proteins on the plasma membrane of rose cells. Plant Physiol. 112, 1261-1271.

Showalter, A.M. (1993) Structure and function of plant cell wall proteins. Plant Cell, 5, 9-23.

Smallwood, M., Martin, H. and Knox, P. (1995) An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins. Planta, 196, 510-522.

Smallwood, M., Yates, E.A., Willats, W.G.T., Martin, H. and Knox, P.J. (1996) Immunochemical comparison of membrane- associated and secreted arabinogalactan-proteins in rice and carrot. Planta, 198, 452-459.

St6hr, C., Snogerup, L., Pennell, R.I. and Kjellbom, P. (1996) An agarose gel electrophoresis method for the separation of arabinogalactan proteins. Plant J. 10, 943-948.

Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. ScL USA, 76, 4350-4354.

Tsumuraya, Y., Mochizuki, N., Hashimoto, Y. and Kov~c, P. (1990) Purification of an exo-~-(1-3)-D-galactanase of Irplex lacteus (Polyporus tulipiferae) and its action on arabinogalactan proteins. J. Biol. Chem. 265, 7207-7215.

Varner, J.E. and L/n, L.-S. (1989) Plant cell wall architecture. Cell, 56, 231-239.

Waffenschmidt, S., Woessner, J.P., Beer, K. and Goodenough, U.W. (1993)Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas. Plant Cell, 5, 809-820.

Wu, G., Short, B.J., Lawrence, E.B., Levine, E.B., Fitzsimmons, K.C. and Shah, D.M. (1995) Disease resistance conferred by expression of a gene encoding H202-generating glucose oxidase in transgenic potato plants. Plant Cell, 7, 1357-1368.

Yariv, J., Rapport, M.M. and Graf, L. (1962) The interaction of glycosides and saccharides with antibody of the corresponding phenylazo gtycosides. Biochem. J. 85, 383-388.

oxidative cross-linking of plasma membrane arabinogalactan proteins

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