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Journal of Experimental Botany, Vol. 48, No. 316, pp. 1925-1933, November 1997 Journal of Experimental Botany Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings Remigio Lanubile, Gabriella Piro and Giuseppe Dalessandro1 Dipartimento di Biologia, Universita di Lecce, via prov.le Lecce-Monteroni, 1-73100 Lecce, Italy Received 9 May 1997; Accepted 18 June 1997 Abstract The in vivo effect of Brefeldin A (BFA) on the synthesis and transport of cell wall polysaccharides and proteins in the roots of pea seedlings (Pisum sativum L. cv. Alaska) was investigated. BFA (10 //gml" 1 ) inhibited the synthesis of cell wall matrix polysaccharides by approximately 43%. Under the same conditions, cellulose synthesis was inhibited by approximately 77%. The percentage of incorporation of L-[U-14C]leucine and L-[U-14C]proline into cytosolic, membrane and cell wall proteins was only slightly changed in the presence of BFA. In addition, the drug did not change the pattern of newly synthesized proteins in the three fractions as judged by SDS-PAGE fluorography. Double labelling of proteins and cell wall polysaccharides confirmed the above reported data. All these results showed that the synthesis and transport of proteins to the cell wall was only slightly affected by BFA under similar conditions to those which brought about a strong inhibition of the synthesis of matrix and cellulosic polysaccharides. BFA had no effect on the activity of membranebound and digitonin-solubilized mannan and glucomannan synthase isolated from the third internode of pea seedlings. This would exclude an effect of BFA at the level of the catalytic site of the synthases. The inhibition of polysaccharide synthesis by the drug was rapidly eliminated after its removal. It is concluded that the effect of BFA on the biosynthesis of cell wall polysaccharides could be caused by an interaction of the drug with the topological organization of the synthase complexes in the membranes. This effect would precede the action of the drug at the level of vesicle transport to the walls. 1 Key words: Brefeldin A, cell wall polysaccharides (synthesis and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport). Introduction The Golgi bodies of both animal and plant cells are dynamic organelles which play a key role in processing, maturation and sorting of newly synthesized proteins, glycoproteins and proteoglycans. These are transported to specific membrane domains or they are secreted from the cell. The Golgi bodies are also involved in the recycling of receptors during endocytosis. In plant cells the Golgi apparatus is the site of synthesis and packaging of cell wall matrix polysaccharides such as pectins and hemicelluloses (Northcote, 1985). Biochemical and immunocytochemical studies have shown that the synthesis of matrix polysaccharides occurs in different cisternae of the Golgi stacks before they are packaged into secretory vesicles. Vesicles take part in the intra-Golgi transport of the polymers in a cis-to-lrcins Golgi direction (Zhang and Staehelin, 1992; Staehelin and Moore, 1995). Cellulose is polymerized at the plasma membrane. The enzymic complexes required for the synthesis of pectins, hemicelluloses, and cellulose are synthesized, at least in part, on the endoplasmic reticulum and then transported and sorted to the Golgi cisternae. Cellulose synthase complex is transported to the plasma membrane by secretory vesicles. Thus, the dynamic structural and functional organization of the plant cell wall during growth and differentiation depends on the activities of the enzymes associated with the endomembrane system. Brefeldin A (BFA), a macrocyclic hydrophobic lactone isolated from a variety of fungi (Harri et ai, 1963; Betina, To whom correspondence should be addressed. Fax: +39 832 320626. E-mail: [email protected]. Abbreviations : ARF1, ADP-ribosylation factor 1; BFA, Brefeldin A. Oxford University Press 1997 1884 Miller et al. v_ c -;.-». _.. .i'v Fig. 2. Electron micrograph of a growing root hair of Equisetum hyemale showing Golgi vesicles in the hemisphere of the tip and Golgi bodies (GB) subapically. Bar= 1 ^m. towards the edge of the pollen tube when nearing the vesicle-rich region and are absent from the vesicle-rich region proper (Miller et al., 1996). Treatment with caffeine, a substance that greatly diminishes the calcium influx and abolishes the tip-focused calcium gradient (Pierson et al., 1996), causes the actin filaments to redistribute in such a way that they are closer to the tip, thereby shortening the vesicle-rich region. When caffeine is removed, the vesicle-rich region re-establishes its normal size simultaneously with the removal of actin filaments from this area (Miller et al., 1996). For further discussion of pollen tube actin, see Miller et al. (1996). Since pollen tubes and root hairs are both polar growing cells with similar morphology, organelle distribution and cytoplasmic streaming patterns, it would seem logical that the actin distribution within growing root hairs is similar to that of a pollen tube. Future experiments should address this issue. Spectrin-like antigen Animal spectrins are multifunctional molecules that belong to a family of proteins including actin binding protein 1, a-actinin, dystrophin, and fimbrin. These proteins have many binding sites with at least two actin filament binding sites, and binding sites for calcium and calmodulin (Hartwig, 1994). Spectrin and its associated proteins in red blood cells are now thought to provide organizational stability to a cell by controlling integral membrane protein distribution (Bennett and Gilligan, 1993; Devarajan and Morrow, 1996). Based on data from adrenal chromaffin cell secretion Hays et al. (1994) suggest that an increase of cytosolic calcium concentration ([Ca2+]c) dissociates actin from spectrin thereby allowing vesicle fusion. Immunoblot analysis of young tomato leaves detected a 220 kDa protein labelled with human erythrocytespectrin (Michaud et al., 1991), and Faraday and Spanswick (1993) detected a 230 kDa protein with antispectrin in the purified plasma membrane fraction of rice roots. In addition, De Ruijter and Emons (1993) detected spectrin-like epitopes in several tissues of maize and carrot, predominantly at the plasma membrane of cells growing along their whole length. This spectrin-like epitope is also localized at the tip of growing root hairs (Fig. 3), which is comparable to its localization at the tips of tobacco pollen tubes (Derksen et al., 19956) and the tips of growing hyphae of Saprolegnia ferax (Kaminskyj and Heath, 1995). Even though the nature of the plant epitope found in root hairs is still not known, its location suggests a role in exocytosis. Calcium gradient External Ca2+ ions at the appropriate concentration and a Ca2+ influx are required to elicit exocytosis in Arabidopsis root hairs (Schiefelbein et al., 1992). These authors observed a net influx of Ca2+ at the tips of growing root hairs with a calcium selective vibrating probe, whereas no influx was found at the sides of the hair nor into non-growing hairs. Selective vibrating probe analysis indicated Ca2+ currents only in the tips of growing Sinapis root hairs (Herrmann and Felle, 1995; Felle and Hepler, 1997), which responded to changes in external concentrations of calcium by transient growth differences rather than an altered steady-state growth rate (Herrmann and Felle, 1995). The [Ca2+]c increased in the apical area in the presence of increased external calcium and decreased when the external calcium concentration was lowered (Felle and Hepler, 1997). In the latter study non-growing hairs responded to changes in external calcium as well, but the [Ca2+]c increase was uniform 1926 Lanubile et al. 1992), has been widely used in animal cells to investigate the transport of proteins and glycoproteins along the secretory pathway and also the structure and function of the Golgi apparatus. It causes numerous effects such as (a) the inhibition of vesicular transport of membranes and secretory proteins between the endoplasmic reticulum and Golgi and intra-Golgi transport (Misumi et al., 1986; Oda et al., 1987); (b) the rapid disassembly of the Golgi apparatus and the redistribution of Golgi proteins and lipids into the endoplasmic reticulum by a mechanism which requires energy and microtubules (Fujiwara et al., 1988; Domse/a/., 1989; Lippincott-Schwartz e? al., 1990); (c) the release of the non-clathrin coated protein jS-COP from the Golgi apparatus (Donaldson et al., 1990); (d) changes in the morphology and function of other components of the endomembrane system such as endosomes, lysosomes and the trans Golgi network (TGN) (Lippincott-Schwartz et al., 1991; Klausner et al., 1992); (e) the stimulation of the synthesis of sphingomyelin, glucosylceramide and cholesterol ester and at the same time the inhibition of the synthesis of cholesterol, triacylglycerol and phosphatidyl-choline (Kallen et al., 1993); (f) the alteration of synthesis and processing of O- and TV-linked glycans (Chawla and Hughes, 1991; Collins and Mottet, 1992). BFA appears to cause most of its effects at the Golgi apparatus by inhibiting the activation of ADP-ribosylation factor 1 (ARF1) catalysed by the Golgi-associated GTP/GDP exchange protein. This prevents the binding of ARF1 and coatomer, a cytosolic coat protein complex, to Golgi membranes (Orci et al., 1991; Donaldson et al., 1992; Helms and Rothman, 1992). Thus ARF1, or an ARF-activating protein (Randazzo et al., 1993), seem to be the key BFA-sensitive component required for COPs binding to Golgi membranes. All morphological and physiological effects of BFA disappear when the drug is removed from the cells. Recently, BFA has been used to elucidate the morphological and functional changes of the plant Golgi apparatus as well as other components of the endomembrane system. Ultrastructural (Satiat-Jeunemaitre and Hawes, 1992; Rutten and Knuiman, 1993; Yasuhara et al., 1995; Gorbatenko and Hakman, 1995), immunocytochemical (Satiat-Jeunemaitre and Hawes, 1993; Henderson et al., 1994; Satiat-Jeunemaitre et al., 1996) and biochemical (Holwerda et al., 1992; Driouich et al., 1993; Jones and Herman, 1993; Gomez and Chrispeels, 1993; Schindler et al., 1994; Kunze et al., 1995) investigations on different plant materials have confirmed that many aspects of BFA effects on plant cells are similar to those reported in animal cells. In particular, secretion of soluble proteins to the vacuole (Driouich et al., 1993; Holwerda et al., 1992; Jones and Herman, 1993; Gomez and Chrispeels, 1993) and matrix polysaccharides such as pectins and hemicelluloses to the wall (Driouich et al., 1993) are inhibited by BFA whereas the drug does not interfere with the transport of the tonoplast integral protein (aTIP) (Gomez and Chrispeels, 1993). These results have indicated that proteins may follow different pathways to the same destination. It has also been reported that low concentrations of BFA modify secretion and glycosylation of secretory proteins without any breakdown of the Golgi stacks (Driouich et al., 1993; Gomez and Chrispeels, 1993; Kimura et al., 1993). In the present study the in vivo effects of BFA on the synthesis and transport of cell wall polysaccharides and proteins in the roots of pea seedlings (Pisum sathntm L. cv. Alaska) were investigated. The results obtained demonstrate that BFA rapidly inhibits the biosynthesis of cellulose and matrix polysaccharides whereas the synthesis and transport of proteins to the cell wall remain unchanged. Materials and methods Chemicals and radiochemicals Brefeldin A, GDP-D-mannose, GDP-D-glucose and all other chemicals were purchased from Sigma Chemical Co., St Louis, Mo., USA. D-[U-14C]glucose (specific activity 10.5 GBq mmol" 1 ), L-[U-14C]proline (specific activity 9.32 GBq mmol" 1 ), L-[U-14C]leucine (specific activity 11.7 GBq mmol"'), L-[4,53 H]leucine (specific activity 3.15 TBq mmol" 1 ), L-[35S]methionine (specific activity >37 TBq mmol" 1 ), GDP-[U- 14 C]glucose (specific activity 8.40 GBq mmol" 1 ), GDP-[U- 14 C]mannose (specific activity 11.2 GBq mmol" 1 ) were from Amersham International, Amersham, Bucks, UK.. Plant material and radiolabellmg expenments Seeds of Pisum sativum L. cv. Alaska were soaked in running tap water (8 h), planted in damp vermiculite and grown for 2 d at 25±1°C in darkness. Using a dim green light, uniform seedlings having primary roots 20-22 mm long were carefully washed in sterile bidistilled water and incubated with or without different concentrations of BFA in the presence of the radioactive tracers as specified for each experimental condition. The uptake of the radioactive tracer represents the difference between the initial radioactivity of the incubation mixture and the radioactivity remained at the end of incubation period of the seedlings. Radioactive cell wall polysaccharide labelling Three pea seedlings were incubated in water with or without BFA in the presence of 4 ^Ci of r>[U- 14 C]glucose at 25 "C, in the dark, as specified for each experiment in tables and figures. At the end of the radiolabelling period, the roots were rapidly washed with an ice-cold unlabelled tracer solution and the root cap removed. Starting from the root apex, segments 1 cm long were cut from each root and homogenized with 2 ml Tenbroeck tissue grinder in 10 mM sodium-phosphate buffer pH 7.2, containing 10 mM imidazole, 1 mM benzamidine, 5 mM 6-amino-esanoic acid, 10 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The final volume of the homogenate was measured and aliquots were counted for radioactivity in order to obtain the total amount of the radioactive material present in each homogenate. The homogenates were centrifuged at 800 x g for 10 min at 2 =C in a model J2 21 centrifuge, rotor head J-20 (Beckman Instruments, Inc., BFA: cell wall and protein synthesis Palo Alto, California, USA). The pellets, consisting of cell walls, were incubated in 0.05% sodium deoxycholate for 2 h, at 20 °C and then washed with the homogenization buffer (twice) and acetone (twice) to obtain purified cell walls. The 800 xg supernatant was centrifuged at lOOOOOxg for 60 min at 2°C in a Beckman preparative ultracentrifuge (model L8-55, rotor head SW 55) to obtain a supernatant (100000 x g SN) and a pellet consisting of membrane and organelles. Each pellet was suspended in 75% ethanol containing 50 g l " 1 glucose and centrifuged at 8000 x g for 5 min at 2 °C (five times) to remove all free sugars. This fraction was the membrane fraction. Purified cell walls and membranes were hydrolysed with 3 N HC1 in sealed tubes at 120°C, for 1 h. The hydrolysates were dried, resuspended in water and counted for radioactivity. The cell wall residue was dissolved in 3% (w/w) H 2 SO 4 and hydrolysed at 120 °C, for 1 h. The hydrolysate was neutralized with a 15% (v/v) solution of methyl-di-H-octylamine in chloroform (excess of amine was removed by washing with five changes of chloroform), taken to dryness, resuspended in water and counted for radioactivity (cellulose). The lOOOOOxg SN was treated with 60% ethanol (final concentration) and centrifuged in a J2-21 centrifuge, rotor head J-20 for 10 min at lOOOOxg. The pellet was washed five times with 60% ethanol (v/v). The pellet and aliquots of the lOOOOxg supernatant were counted for radioactivity in order to determine radioactive soluble polysaccharides and cytosolic pool, respectively. Radioactive protein labelling Six pea seedlings were incubated in water with or without 10 jig ml" 1 BFA for 1 h, at 25°C, in the dark. During the last 30 min of incubation 8 ^Ci of L-[U-14C]proline, 8 ^Ci of L-[UI4 C]leucine or 750 /nCi of L-[35S]methionine were added to the incubation medium. At the end of the radiolabelling period, membranes and purified cell walls were isolated. To evaluate the radioactivity incorporated into cytosolic proteins, the lOOOOOxg SN was concentrated (Centricon 10, Amicon) and the proteins were precipitated with 80% acetone at — 20 °C overnight. Membrane proteins were obtained from membranes depleted of lipids as described by Dalessandro et al. (1986). The proteins were precipitated with 80% acetone (final concentration) at — 20 °C overnight. Purified cell walls were resuspended and agitated in phenol: acetic acid:H 2 0 (2/1/1, w/v/v) at 70°C for 30min (Fry, 1988) to extract non-covalently/ ionically bound cell wall proteins (PAW extract). After centrifugation (8700 xg, 10 min) the proteins present in the supernatant were precipitated with 0.1 M ammonium acetate in methanol over night. The 8000 x g pellet, consisting of cell wall and covalently bound proteins, was hydrolysed with 3 N H O in sealed tubes at 120°C, for l h (HC1 hydrolysate). PAW extract and H O hydrolysate were dried, resuspended in water and counted for radioactivity. Cytosolic, membrane and cell wall proteins were analysed by SDS-PAGE electrophoresis (Laemmli, 1970) on a 13% polyacrylamide gel. Protein bands were stained with Coomassie brilliant blue R-250. Labelled proteins were detected by fluorography on Hyperfilm-/3-max (Amersham). Double radiolabelling of proteins and cell wall polysaccharides Six pea seedlings were incubated in water with or without 10/xgmr 1 BFA for 1 h, at 25 °C, in the dark. During the last 30 min of incubation, 8 fid of r>[U-14C]glucose and 100 ^Ci of L-[4,5-3H]leucine were simultaneously added to the medium. After radiolabelling, the cytosolic pool, soluble polymers, 1927 membrane and cell wall polysaccharide and proteins were isolated. Mannan and glucomannan synthesis The effect of BFA (10 and 50/igml" 1 for 30 min) on the in vitro synthesis of mannan and glucomannan was studied by using membrane-bound and digitonin-solubilized enzymes isolated from the third internode of pea seedlings. Enzymic preparations, incubation procedure and isolation of radioactive polymers were performed as reported by Piro et al. (1993). Enzymic assays were performed by using membrane-bound or digitonin-solubilized enzymes preincubated or not in the presence of BFA. Radioactivity counting procedure Radioactivity counting procedure was performed as described by Piro et al. (1993), using a 1217 Rack Beta liquid scintillation spectrometer (LK.B). Counting efficiency was approximately 90%. Results All experiments used 2-d-old pea seedlings which were incubated for short times with and without BFA in the presence of D - [ U - 1 4 C ] glucose, L-[U-14C]leucine, L - [ U 14 C]proline or both D-[U-14C]glucose and L - [ 4 , 5 - 3 H ] leucine. Segments (1 cm) of decapitated roots were used to estimate the incorporation of radioactive sugars and amino acids into cell wall polysaccharides and proteins. Incorporation data are expressed as percentages of the total radioactivity measured in the homogenate of the root segments. BFA at 10 and 20 jig m l " 1 inhibited D-[U- 1 4 C]glucose uptake in pea seedlings by approximately 16% and 50%, respectively, up to 1 h of incubation (Fig. 1A). A more pronounced inhibition by BFA was observed in the total amount of radioactive material found in the homogenates obtained from decapitated root segments isolated from BFA-treated seedlings (Fig. IB). The incorporation of radioactive sugars into matrix polysaccharides was inhibited by BFA (10/^g ml" 1 ) by approximately 17% and 30% after 30 and 60 min, respectively. BFA at 20 jig ml" 1 markedly inhibited the incorporation of radioactive sugars into matrix polysaccharides from 15 min of incubation (Fig. 1C). At 10 and 20 jig ml" 1 of BFA, the biosynthesis of cellulose, expressed as a percentage of radioactive glucose incorporated into the cellulose, was drastically inhibited after 15 min of incubation. BFA at 10 jig ml" 1 inhibited cellulose synthesis more rapidly than matrix polysaccharides. To test how BFA affects the synthesis and transport of cell wall polysaccharides to the wall, pea seedlings were incubated for 1 h with and without BFA (10 jig ml" 1 ) and after 30 min of incubation, D-[U- U C]glucose was added in the medium. In these experimental conditions the inhibitory effect of BFA on the synthesis of cell wall polysaccharides had already started before the addition 1928 Lanubile et al. 100 Table 1. Effect of BFA on radioactive lOOOOOxg SN and radioactive polvsaccharides found in membrane and purified cell wall (A) D-[U- 14 C]glucose uptake 80- The subcellular fractions were isolated from 1 cm long root segments obtained from three pea seedlings incubated in water with or without lOngml" 1 BFA for 1 h, at 25°C, in the dark. During the last 30min of incubation, 4 ^Ci of D-[U-'4C]glucose were added in the medium. Incorporation is expressed as a percentage of the total amount of the radioactivity measured in the homogenate. The data are from one representative experiment of three. GO40 • 20- Radioactivity (% of the homogenate) 50 • (B) homogenate 40 • lOOOOOxgSN Membrane Cell wall Matrix polysacchandes Cellulose Total radioactivity of the homogenate (kBq) 302010 • H2O lO^gml" 1 BFA 73 9 1 8 87 0 1.7 16.6 7.7 9.5 1.8 20.4 20.1 10 ' (C) matrix polysaccharidcs 8 6 • 4 • 2- 10 • (D) cellulose 8 6 4 ' 2 • 15 30 45 I 60 Incubation time (min) Fig. 1. Effect of BFA on: uptake of D-IU-^Clglucose by pea seedlings (A), radioactive material present in the homogenate (B), radioactive sugars incorporated into matrix polysaccharides (C) and cellulose (D) in 1 cm long pea root segments. 2-day-old pea seedlings (three) were incubated in water with or without BFA (10 and 2 0 n g m l " 1 ) in the presence of 4^Ci of D-[U-14C]glucose for 15, 30 and 60 min. At the end of incubation period, the seedlings were rapidly washed in a solution of cold glucose and depleted of root cap. Starting from the root apex, a segment 1 cm long was cut from each root and processed to analyse the amount of radioactivity present in the homogenate and incorporated into cell wall polysaccharides. ( • ) Water, (A) BFA 10 fig m l " ' , ( • ) BFA 20 ^g m l " ' . The data are from one representative experiment of three. of radioactive tracer. Table 1 shows that the percentage of radioactive sugars incorporated into cellulose and matrix polysaccharides in both membranes and cell walls was inhibited in root segments isolated from BFA-treated pea seedlings. The inhibitory effect of BFA on the matrix polysaccharides in membranes and cell walls was 6% and 43%, respectively. Under the same conditions, cellulose was inhibited by approximately 77%. The lack of any accumulation of radioactive matrix polysaccharides in membranes suggested that an inhibition of matrix polysaccharide synthesis occurred before an inhibition of their transport to the wall. Pea seedlings were incubated for 1 h with BFA ( l O ^ g m l " 1 ) , and during the last 30 min of incubation, L-[U-14C]proline or L-[U-14C]leucine was added to the medium. BFA inhibited the uptake of L-[U-14C]proline and L-[U-14C]leucine by approximately 60% and 50%, respectively. A similar inhibition was found for the total amount of radioactive material present in the homogenates. However, the percentage of incorporation of L-[U14 C]leucine and L-[U-14C]proline into cytosolic, membrane and cell wall proteins was only slightly changed in the presence of BFA (Table 2). In particular the percentage of L-[U-14C]leucine and L-[U-14C]proline incorporated into both non-covalently/ionically bound (PAW extract) and covalently bound (HC1 hydrolysate) cell wall proteins was slightly stimulated. The synthesis and transport of newly synthesized proteins to the cell wall was only slightly affected by BFA under similar conditions to those which brought about a strong inhibition of the synthesis of matrix and cellulosic polysaccharides. Confirmation that the biosynthesis of cell wall polysaccharides was inhibited by BFA without affecting the synthesis and transport of cell wall proteins was obtained when pea root seedlings were incubated for 1 h with BFA and during the last 30 min D-[U-14C]glucose and L-[4,5-3H]leucine were added simultaneously to the incubation medium. The percentage of [ 3 H]material BFA: cell wall and protein synthesis 4 1929 14 Table 2. Effect of BFA on the incorporation of L-[U-' CJprohne or L-[U- C] leucine into cytosolic, membrane and cell wall proteins extracted from pea root segments Cytosolic, membrane and cell wall proteins were isolated from 1 cm long root segments obtained from six pea seedlings incubated in water with or without 10/igml' 1 BFA for 1 h, at 25 °C, in the dark. During the last 30 min of incubation 8 MCi of L-[U-14C]proune or 8/xCi of L-[U-14C]leucine were added to the medium. Incorporation is expressed as a percentage of the total amount of radioactivity measured in the homogenate. The data are from one representative experiment of three. Proteins Radioactivity (% of the homogenate) L-[U-14C]proline Cytosolic Membrane Cell wall PAW extract HC1 hydrolysate Total radioactivity of the homogenate (kBq) L-[U-14C]leucine H2O 10 Mg ml" 1 BFA H2O 10 y.% ml" 1 BFA 18.4 12.4 17.4 11.8 19.0 11.9 14.3 12.0 1.8 0.2 2.2 0.4 3.1 0.6 3.4 0.8 51.3 14.7 60.3 22.9 present in the cytosolic pool and the percentage of radioactive leucine incorporated into soluble, membrane and cell wall proteins was only slightly affected by BFA (Table 3) but the incorporation of [14C]sugars into cell wall polysaccharides and into the oligosaccharide sidechains (much less) of cell wall glycoproteins was inhibited. In addition, the percentage of radioactive polymers found in membranes (matrix polysaccharides and the oligosaccharide side-chains of glycoproteins and glycolipids) remained almost unchanged in BFA-treated seedlings. The percentage of radioactive sugars incorporated into both soluble polysaccharides and glycoproteins which co-precipitated with cytosolic proteins was stimulated by BFA. The pattern of newly synthesized cytosolic, membrane and cell wall proteins isolated from BFA-treated and untreated pea-root seedlings incubated in the presence of L-[35S]methionine is shown in Fig. 2. BFA does not change the pattern of proteins in the three fractions. The proteins extracted from purified cell walls of BFA-treated and untreated roots remained qualitatively and quantitatively the same. This confirmed that the synthesis and transport of newly synthesized proteins into the wall were not changed by BFA. Similar results were obtained when L-[35S]methionine was substituted from L - [ U - 1 4 C ] leucine (data not shown). BFA (10 and 50 fig ml" 1 ) on membrane-bound and digitonin-solubilized mannan and glucomannan synthase isolated from the third internode of pea seedlings did not change the specific activity of the synthases (Table 4) even when the membrane-bound and the digitoninsolubilized enzymes were pretreated for 30 min with BFA before the addition of substrates (data not shown). The inhibition of polysaccharide synthesis produced in pea-root seedlings by BFA was eliminated after its removal (Fig. 3). The synthesis of cellulose recovered rapidly from the BFA effect, reaching 94.5% and 100% of the control level after 20 and 30 min, respectively. The recovery of the synthesis of matrix polysaccharides by Table 3. Effect of BFA on the incorporation of D-[U-l4CJglucose and L-/4,5-3H]leucine into soluble, membrane and cell wall polymers (proteins and polysaccharides) extracted from pea root segments 1 cm long pea root segments were isolated from six pea seedlings incubated in water with or without lOfig ml" 1 BFA for 1 h, at 25°C, in the dark. During the last 30 min of incubation 8 ^C\ of r>[U-14C]glucose and 100 ^Ci of L-{4,5-3H]leucine were simultaneously added to the medium. Incorporation is expressed as a percentage of the total amount of the radioactivity measured in the homogenate. The data are from one representative experiment of three. Radioactivity (% of the homogenate) tO/xgml-'BFA H2O 14 D - [ U - C ] glucose Cytosolic pool Soluble polymers Membrane Cell wall PAW-extract (proteins) HC1 hydrolysate (matrix polymers) H 2 SO 4 hydrolysate (cellulose) Total radioactivity of the homogenate (kBq) 3 L-{4,5- H] leucine D-[U-'4C]glucose L-[4,5-3H] leucine 40.9 I.I 7.2 23.5 3.7 6.0 45 0 1.9 7.5 25 2 4.1 6.1 2.1 6.8 5.9 2.2 0.3 1.7 3.7 3.1 2.2 0.2 31.9 167.0 25.0 82.3 1930 Lanubile et al. membrane 200.0 97.4 69.0 kDa — — — 46.0 — 30.0 — cytosol 14.3 H2O BFA H2O BFA H2O BFA Fig. 2. Pattern of cell wall, membrane and cytosolic proteins extracted from BFA-treated and untreated pea root seedlings. Cell wall, membrane and cytosolic proteins were isolated from 1 cm long root segments obtained from 30 pea seedlings incubated in water with or without l O ^ g m r 1 BFA for 1 h, at 25 °C in the dark. During the last 30min of incubation 750 ^Ci of L-[35S]methionine were added in the medium The amount of radioactive proteins on each lane is approximately 250000 dpm. Standard protein molecular-weight markers (kDa) are on the left. BFA although slower than that of cellulose was established between 20 and 30 min. Discussion The results show that in pea root seedlings BFA rapidly and markedly inhibits the synthesis of both cellulose and cell wall matrix polysaccharides without altering the synthesis and transport of newly synthesized proteins into the walls. Therefore under these experimental conditions the inhibitory effect of BFA on cell wall polysaccharides cannot be due to an inhibition of vesicular traffic to the wall, but to an indirect or direct effect on the activity of polysaccharide synthase complexes located at the membranes. Driouich et al. (1993) working with sycamore suspension-cultured cells showed that, although the newly synthesized cellular proteins in sycamore cells were only slightly inhibited by BFA, their secretion into the culture medium was inhibited. The incorporation of [3H]xylose from UDP-D-[3H]xylose and [3H]fucose into the hemicelluloses extracted from the cell wall of BFA-treated sycamore cells was also inhibited. The close correlation between the inhibitory effect of BFA on protein secretion into the culture medium and the reduced amount of radioactive sugars incorporated into the hemicelluloses extracted from the walls was taken as proof that BFA brought about its effects by an inhibition of the secretory pathway. However, using the same experimental conditions they showed that the incorporation of [ 3 H]mannose, [3H]xylose from UDP-D-[3H]xylose and [3H]fucose into the oligosaccharide chains of glycoproteins secreted within the cell or to the culture medium was differentially inhibited by BFA. They suggested that the drug acted on the synthesis and/or processing of N-linked glycans. However, Driouich et al. (1993) gave no explanation for the fact that the amount of newly synthesized glycoproteins secreted into the culture medium calculated as percentage with respect to newly synthesized cellular glycoproteins remained unchanged or was even stimulated in BFA-treated sycamore cells compared to that of control cells. Thus, if the inhibitory effect of BFA was mainly due to an inhibition of the vesicular traffic, as postulated by Driouich et al. (1993), it is difficult to explain why BFA did not alter but even stimulated the secretion of unglycosylated or incorrectly glycosylated proteins into the culture medium. The results of this study show that, in pea root seedlings, cellulose and cell wall matrix polysaccharides are inhibited by BFA under experimental conditions in which it is possible to verify that (a) the synthesis and transport Table 4. Effect of BFA on membrane-bound and digitonin-solubilized mannan and glucomannan synthase isolated from the third internode of pea seedlings Mannan- and glucomannan-synthase activity was measured as the amount of either [14C]mannose or [14C]glucose incorporated in the [14C]polymer. For the mannan-synthase activity the reaction mixture contained: 0 20 nmol of GDP-r>[U-14C]mannose (approximately 1500 Bq), 2.5 nmol of GDP-D-mannose, BFA (10 or 50^gml~') and membrane enzymic preparation (180^g of protein) or digitonin-solubilized enzymes (50^g of protein). For the glucomannan-synthase activity the reaction mixture contained: 0.27 nmol of GDP-D-[U-'4C]glucose (approximately 1500 Bq), 1.0 nmol of GDP-D-glucose, 5.0 nmol of GDP-D-mannose, BFA (10 or 50 ^g ml" 1 ) and membrane enzymic preparation (180 ^g of protein) or digitonin-solubilized enzymes (50^g of protein). Reaction time at 27 °C was 30 min. The data are from one representative experiment of three. BFA Specific activity (nmol min" 1 mg" 1 of protein) Membrane-bound enzyme 50 jig ml Digitonin-solubilized enzyme Mannan-synthase Glucomannan-synthase Mannan-synthase Glucomannan-synthase 3.80 3.70 3.75 0.40 0.45 0.38 1.90 2.00 1.85 0.22 0.20 0.24 BFA: cell wall and protein synthesis IWWIMUWmmatM 0 incubation time (min) ^ 1 60 I I 1520 I 30 I 60 recovery time (min) H2O BFA 10 |ig ml-1 D-[U-14C] glucose 15 20 30 Recoverytime(mm) Fig. 3. Recovery of the biosynthesis of cell wall polysacchandes from pretreatment with BFA in pea root seedlings. 1 cm long pea root segments were isolated from 3 pea seedlings incubated with or without 10 ^g ml" 1 BFA for 60 min, in the dark at 25 °C. The recovery from the effect of BFA was studied by transferring seedlings in water for 15, 20, 30, and 60 min and adding 4 ftCi of D-[U-14C]glucose as radioactive tracer in the last 10 min of incubation as reported in the scheme. (A) Cellulose, ( • ) matrix polysaccharides. The data are from one representative experiment of three. of newly synthesized proteins into the wall was not affected by the drug (Tables 2, 3); (b) the patterns of newly synthesized cytosolic, membrane-bound and cell wall proteins isolated from BFA-treated and control roots were similar (Fig. 2); (c) the inhibition of cellulose synthesis started at a concentration of BFA which did not affect the synthesis and transport of cell wall matrix polysaccharide into the wall. These data show that the inhibitory effect of BFA on cell wall polysaccharide synthesis started before any effect on the vesicular traffic of proteins to the walls. In support of this idea, Kimura et al. (1993) have demonstrated that the application of 1931 BFA to suspension-cultured rice cells inhibited the synthesis of O-linked saccharide chains (recognized by peanut lectin (PNA) and Ulex europaeus lectin-I (UEA-I)) of Golgi membrane glycoprqteins. It was also shown that both the protein composition of Golgi membrane and the intracellular transport of a-amylase molecules were not affected by BFA. It has also been reported that as a consequence of BFA treatment an altered O- and Nglycosylation occurred in mammalian and plant cells (Chawla and Hughes, 1991; Collins and Mottet, 1992; Sampath et al., 1992; Driouich et al., 1993; Gomez and Chrispeels, 1993). The cellulose synthase complex of the plasma membrane is not only involved in the synthesis of j3-glucan chains but is also implicated in their association into micro- and macro-fibrils. The complex has catalytic subunits for the synthesis of /3-glucan chains and a highly organized number of structural and regulatory proteins which bring about the crystallisation of /S-glucan chains into cellulose I (Northcote, 1991; Delmer and Amor, 1995). It was found that BFA, given simultaneously with a radioactive tracer, rapidly inhibited cellulose synthesis (Fig. ID). It was also observed that in vitro BFA did not change the activity of the membrane-bound and digitoninsolubilized mannan and glucomannan synthase isolated from the third internode of pea seedlings. In animal systems also, BFA does not affect the sugar nucleotide transporters or the glycosyltransferases (Misumi et al., 1986). All the evidence indicates that the inhibitory effect of BFA on both cellulose and matrix polysaccharides formation is not at the catalytic site of the synthases. Little is known about the structural and functional organization of polysaccharide synthase complexes in membranes but it is probable that, similar to the cellulose synthase complex, different proteins such as donor substances, transferases and acceptor molecules are involved in the synthesis of cell wall matrix polysaccharides. These proteins must interact very closely with the phospholipids of membranes since in vitro membrane-bound polysaccharide synthases lose their activity very rapidly in the presence of phospholipases (Piro et al., 1993). Since BFA is known to be a lipophilic molecule which interacts with membranes, the effect of BFA on the biosynthesis of cell wall polysaccharides could be caused by an interference of the drug with the topological organization of the synthase complexes in the membranes. This effect would precede the action of the drug at the level of vesicle transport of proteins to the walls. This would explain the differential sensitivity to the drug, at the Golgi apparatus, toward the synthesis of cell wall polysaccharides and the machinery of vesicular transport of proteins to the wall. A differential action of BFA on membrane compartments has been reported in animal and plant cells (Hunziker et al, 1991; Pelham, 1991; Gomez and Chrispeels, 1993). The synthesis of glycosaminoglycan chains occurs in 1932 Lanubile et al. most eukaryotic cells (Hardingham and Fosang, 1992). In condrocytes of chondrosarcoma cells from the swarm rat, the disruption of vesicular traffic by BFA rapidly inhibited the synthesis of chondroitin sulphate, the major structural components of proteoglycan aggrecan. However, the hyaluronan synthase complex, at the plasma membrane, was much more stable and hyaluronan synthesis continued at the same rate up to 8 h. The recovery of chondroitin sulphate synthesis from the action of BFA after its removal occurred in the presence of cycloheximide so that its synthesis probably reassembled from previously existing proteins (Calabro and Hascall, 1994a, b). These results, in contrast to our results, indicated that BFA had no effect on the glycosyltransferases involved in the chondroitin sulphate and hyaluronan synthesis. It must be noted, however, that the morphology and function of the Golgi apparatus differ between plant and animal cells. It is also established, as has already been indicated, that the action of BFA varied not only with the concentration of the drug applied to the tissue but even between different species of plants. Differences between the action of BFA on plant and animal cells might therefore be expected. It is possible that a more complex organization of polysaccharide synthases including the glycosyltransferases, is present on plant membranes compared to that of animal cells. No polysaccharide synthases have been purified and characterized from higher plants whereas several glycosyltransferases have been purified and characterized in animal systems (Paulson and Colley, 1989; Kleene and Berger, 1993). 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