Rhamnogalacturonide Tetrasaccharides lsolated … Rhamnogalacturonide Tetrasaccharides lsolated from Semi-Retted Flax Fibers Are Signaling ... PI, proteinase inhibitor; ... heated

Plant Physiol. (1997) 115: 793-801

Two Rhamnogalacturonide Tetrasaccharides lsolated from Semi-Retted Flax Fibers Are Signaling Molecules in

Rubus fruticosus L. Cells

Elisabeth Dinand, Cérard Excoffier, Yvette Liénart, and Michel R. Vignon*

Centre de Recherches sur les Macromolécules Végétales-Centre National de Ia Recherche Scientifique, Associe 2 I’Université Joseph Fourier, B.P.

Water extraction of semi-retted flax (Linum usitafissimum 1.) fiber bundles yielded a mixture of pectic oligosaccharides and two acidic rhamnogalacturonide tetrasaccharides that were separated by size-exclusion chromatography. One- and two-dimensional nuclear magnetic resonance studies and fast atom bombardment-mass spectrometry experiments indicated that the two tetrasaccharides have a common primary structure, i.e. cw-D-AGalpA(1--22)-(~-1- Rhap(l+4)-c~-~-GalpA-(l-+2)-~-cw,p-Rhap, with a rhamnopyranose as terminal reducing end, and a 4-deoxy-p-i-fhreo-hex-4-enopyranosiduronic acid at the nonreducing end. However, the two tetrasaccharides differ by an acetyl group located at the 0 3 position of the interna1 galacturonic acid residue. These two tetrasaccharides induce the activation of D-glycohydrolases of Rubus fruc- ticosus 1. cells or protoplasts within minutes.

Flax (Linum usitatissimum L.) is an herbaceous plant used in the textile industry because it produces long cellulosic fibers. These fibers originate from differentiated phloem parenchyma cells. They are bound by the middle lamellae, which is particularly rich in pectic polysaccharides, and are arranged in bundles, separated by the cortical parenchyma cells. The traditional way of separating the cellulose fibers from the rest of the plant consists of a dew-retting process, which occurs when the flax straw is spread out and left in the field for severa1 weeks after harvest. During the retting process microorganisms degrade the pectic substances, al- lowing the dissociation of the fibers. This process is dependent on the pectic enzyme production, which itself de- pends on moisture and temperature conditions. Finally, a mechanical treatment (scutching) is required to separate the fibers.

Pectins are important constituents of plant cell walls and major components of the middle lamellae. They have been extensively studied either by sequential extractions and further purifications or by using specific enzyme degrada- tions. Pectins are reported as complex heteropolysacchar- ides composed of homogalacturonan segments interrupted at intervals by ramified regions. Homogalacturonan regions (“smooth regions”) are sequences of (1+4)-a-~- galacturonic acid residues and are described as having a minimum length of 72 to 100 residues (Thibault et al.,

* Corresponding author; e-mail vignon8cermav.cnrs.fr; fax 33-04-76-54-72-03.

793

53, 38041 Grenoble cedex 9, France

1993). Ramified regions (”hairy regions”) contain a rhamnogalacturonan backbone, which consists of a diglycosyl alternating repeating unit -+4)-a-~-GalpA-( 1+2)-cu-~-Rhap- ( 1 4 , with side chains attached through 0-4 of a rhamnosyl residue. These side chains contain neutra1 sugars, mainly Ara and Gal, of varying length and ramification as found in sugar beets (Guillon et al., 1989), hemp (Vignon and Garcia-Jaldon, 1996), mature onions (Redgwell and Selven- dran, 1986), grape berries (Saulnier et al., 1988), apples (Stevens and Selvendran, 1984; Schols et al., 1990, 1995; Renard et al., 1991), carrots (Massiot et al., 1988), and in suspension-cultured cells of sycamore (McNeil et al., 1980; Lau et al., 1985) and rice (Thomas et al., 1989). Flax pectins have been amply characterized by Morvan and co-workers (Morvan et al., 1989, 1990; Davis et al., 1990). Methylated and homogalacturonan pectins are found in the cortex cell walls of flax, whereas the walls of the phloem cells, which are differentiated into fibers, consist of rhamnogalacturonan pectins called RG-I.

In higher plants the accumu!ation of phytoalexins is a well-established self-defense mechanism (Darvill and Al- bersheim, 1984). This defense response is triggered by sugars, termed oligosaccharins or elicitors, which are active in plant signaling. Oligogalacturonides released from plant cell walls either by acid hydrolysis or by treatment with endogalacturonases and endopectate lyases of microbial origin (Spiro et al., 1993) have been identified as elicitors of phytoalexins in different plant systems (Davis et al., 1986b; Davis and Hahlbrock, 1987). Furthermore, these oligomers play a direct role in helping the plant resist disease. Indeed, they are able to induce proteins related to pathogenesis (Davis and Hahlbrock, 1987; Broeckaert and Peumans, 1988; Farmer et al., 1991), to increase the deposition of lignin (Robertsen, 1986), and to produce isoperoxidases (Bruce and West, 1989) and H,O, (Aposto1 et al., 1989). In addition to these effects in plant defense, oligogalacturonides can influence plant growth and development, since they initiate the ripening process (Campbell and Labavitch,

Abbreviations: COSY, correlation spectroscopy; 1D and 2D, one- and two-dimensional, respectively; FABMS, fast atom bombardment mass spectroscopy; AGalpA, 4-deoxy-@-~-threo-hex-4- enopyranosiduronic acid or 4,5-unsaturated D-galacturonic acid; PI, proteinase inhibitor; RG-I, rhamnogalacturonan 1; Rha, rhamnose.

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794 Dinand et al. Plant Physiol. Vol. 11 5, 1997

1991; Melotto et al., 1994), control organogenesis (Tran Thanh Van et al., 1985), and inhibit auxin-dependent pro- cesses (LoSchiavo et al., 1991; Filippini et al., 1992).

Here we report the isolation, purification, and characterization of two pectic oligosaccharides released during the flax-retting process. We also evaluate the possible biological activities of the two isolated rhamnogalacturonides. When exposed to Xubus fruticosus cells or protoplasts these two isolated rhamnogalacturonides promoted within a few minutes increases in ~-glycohydrolase enzyme levels and trypsin inhibitor activity. These responses suggest that these rhamnogalacturonides may serve as signaling molecules in plant defense.

MATERIALS A N D METHODS

Plant Cell Cultures

Suspensions of Rubus fruticosus L. cultures, originally derived from cambial explants from twigs, were grown as described by Hustache et al. (1975).

Size-Exclusion Chromatography Fractionation

Twenty grams of dried, semi-retted, decorticated flax (Linum usitatissimum L.) fiber bundles were extracted twice with 1.5 L of water at room temperature for 2 h. After concentration to 200 mL and centrifugation, the Iiquid was freeze-dried (1.97g). An aliquot (1 g) was applied to a Bio-Gel P-2 column (length, 180 cm; i.d., 4.4 cm; 200400 mesh, Bio-Rad) and eluted with water. The flow rate (110 mL h-l) was controlled by a peristaltic pump (Milton-Roy, Touzart et Matignon, Courtaboeuf, France), and the refrac- tive index was measured with a differential refractometer (R 403, Waters). The large, unresolved fraction eluted just after the void volume was fractionated again on a Bio-Gel P-2 column eluted with a NH,OAc buffer (0.1 M, pH 3.8) and gave three fractions (Fig. 1). The three fractions were collected and desalted by filtration on the same column eluted with water. Fractions 1 and 2 corresponded to compounds 1 and 2. Fraction 3 was isolated and shown by NMR to be a mixture of high-molecular-weight acidic oligosaccharides and neutra1 arabinogalactan oligosaccharides, but their structures were not further investigated.

Preparation of Protoplasts

Cells in the exponential-growth phase (15-18 d after subculturing) were collected by centrifugation (4000g, 5 min), washed with Heller's medium, and resuspended in 50 mM sodium-citrate buffer (pH 5.9) containing 2% SUC, 4 mM EGTA, 3 mM MgCl,, and 0.06 mM CaC1,. For protoplast isolation, 18- to 20-d-old cells (40 g fresh weight) were incubated overnight at room temperature in 300 mL of the growth medium (pH 5.9) supplemented with 0.56 M mannitol, 0.25% (w/v) cellulase (Caylase 345), and 0.01% (w/v) pectinase (Caylase M3), the latter two purchased from Cayla (Toulouse, France). The released protoplasts were filtered through a 100-pm nylon mesh cloth and washed twice with the incubation medium in the absence of cell

wall-degrading enzymes (5008, 5 min). They were then resuspended in 25 mM Bis-Tris/HCl buffer, pH 4.8, containing 0.56 M mannitol, 0.06 M SUC, 1 mM KCI, 1 mM CaCl,, and 6% (w/v) Ficoll 400, centrifuged at 500g for 5 min, and then washed with Bis-Tris/HCl buffer without Ficoll. Pro- toplast yields ranged from 70 to 85% of the initial number of treated cells.

Bioassays

Protoplasts (or cells) (2 X 106) of R. fvuticosus were suspended in 25 mL of buffer (25 mM Bis-Tris/HCl, pH 4.8, 0.56 M mannitol, 0.06 M Suc, 1 mM KC1, and 1 mM CaC1,) on a roller mixer in the presence or absence of various amounts (up to 3 p ~ ) of inducer. Protoplasts (or cells) were harvested at various time intervals by centrifugation (300g, 8 min) at 4"C, before they were subjected to D-

glycohydrolase or proteinase inhibitor extraction. The viability of protoplasts was assessed by the capacity of intact cells to exclude Evans blue indicator.

D-Glycohydrolase Assays

Enzymes were extracted in 50 mM Tris/HCl (pH 7.2) containing 1 M NaCl by homogenizing the protoplasts on ice with a polytron at full speed, 15 times for 45 s, and the extracts were dialyzed and concentrated using ultrafiltration units equipped with a 10-kD molecular-mass cut-off membrane (Ultrafree-TF, Millipore). Laminarinase ([1+3]-P-D- glucanase, EC 3.2.1.39; 3.2.1.84), chitinase (pOly-[1*4]-p-D- glucosaminidase, EC 3.2.1.14), (1+4)-a-D-galacturonase (EC 3.2.1.15), and P-D-xylosidase (EC 3.2.1.37) were assayed in the presence or absence of cycloheximide (1 p ~ ) or actinomycin D (1 F g mL-l). Substrates, e.g. laminarin (31 pg), chitin (25 pg), polygalacturonic acid (25 pg), or p- nitrophenyl P-D-xylopyranoside (1.5 mM), were incubated at 40°C for O to 4 h with crude enzyme extract (1-2 pg of protein based on Bradford determination [Bradford, 19761) in 200 pL of sodium acetate buffer (0.1 M, pH 5.0). The enzyme reaction was stopped by heating at 100°C for 5 min, and laminarinase or chitinase activity was estimated by quantification of the amounts of reducing sugar end groups, according to Somogyi (1952). P-D-Xylosidase activity was determined according to Lee and Zeikus (1993). For each sample, 8 to 10 enzyme assays were performed from four independent elicitation sets and kinetic curves were drawn. The slopes of the linear-regression fit of the data gave enzyme activities. Enzyme activation was expressed as R, de- fined as the ratio of the slopes obtained from treated versus nontreated protoplasts. Blanks (without enzyme and without substrate) were carried out for each sample.

PI Assays

PIs were extracted from elicited or control protoplasts as described by Rickauer et al. (1989), except that they were heated at 85°C for 10 min. The modulation of trypsin activity by PI (0.4-2.4 pg mL-l protein based on Coomassie Plus Reagent [Pierce] protein assays) was carried out using N-a-benzoyl-D-L-Arg p-nitroanilide as a substrate. For each



Two Rhamnogalacturonide Tetrasaccharides as Signaling Molecules 795

sample, 10 to 15 proteinase assays were performed from at least four independent elicitation sets, and kinetic curves were drawn. The slopes of the linear-regression fits of the data gave proteinase activities min-l), and these were expressed in percent of control without PI. Blanks (without substrate, with PI only) were carried out for each sample.

M S

FABMS spectra were obtained on an MS 50 KRATOS- AEI instrument (Manchester, UK). The samples were dis- solved in a glycerol matrice and submitted to Xe (9 kV) bombardment. FABMS spectra in the positive and negative mode were recorded on an R lOlOC mass spectrometer (model2000, Nermag, Rueil-Malmaison, France), equipped with an M Scan Wallis-type gun (8 kV, 20 mA). The molecular weights were determined by carrying out FABMS directly on the underivatized tetrasaccharides.

N M R

NMR spectra were recorded on a spectrometer (model AC300, Bruker, Wissembourg, France) equipped with a 5-mm dual probe in D,O solution (10 mg mL-* ['H] and 20-50 mg mL-l [I3C]). Chemical shifts were expressed in ppm relative to the methyl signal of internal acetone (which was used as standard) and were taken to be 2.10 ('H) and 31.0 ppm (13C) with respect to the signals for Me,Si at 333K ('H) or 303K (13C). 'H-NMR 1D spectra were recorded using 90" pulses (9 ps), 3000-Hz spectral width, 16K data points, 2.7 s of acquisition time, and eight scans. 'H-NMR 2D COSY experiments were recorded using a standard Bruker sequence, a 256 X 1024 points time- domain matrix over 1000 Hz along the F1 and F2 direc- tions, with F1 zero filled to 1024 points. Sine-bell window functions ( r r / 10 phase-shifted) were used in both direc- tions before Fourier transformation. 1D TOCSY experiments were recorded in the inverse mode with a pulse sequence using DANTE selective inversion: 100 pulses of 2.5 ms each and a delay of 1 ms (10 Hz selectivity); eight acquisition scans were performed for each experiment, with the mixing time being adjusted from 10 to 100 ms.

Nuclear Overhauser effect spectra differences were recorded using the standard NOEDIFF Bruker sequence with a composite pulse presaturation: 3.5 s, decoupler power: 0.4 W, with a 30" pulse length (3 ps) and 1,200 scans. I3C-NMR 1D spectra (75.468 MHz) were recorded using 30" pulse length (2 ps), 15,000 Hz spectral width, 16K data points, 0.5 s of acquisition time, and 3,000 scans.

13C-'H shift-correlation 2D experiments were performed using the conventional XHCORRD Bruker sequence, a 64 X 2048 points time-domain matrix, with a spectral width of 1000 Hz ('H) X 4000 Hz ('"C), which, after zero-filling in F1, gave digital resolutions of 7.8 Hz/point (lH) and 3.9 Hz/point ('"C), with a 90" pulse length (6.5 ps), delay times of 1.5 s between each scan, and 1000 acquisition scans.

Controls

Controls consisting of protoplasts incubated in buffer without elicitor were run concomitantly with the elicited samples. Protoplast viability was monitored during the elicitation treatment by withdrawing, at intervals, 100-pL aliquots from protoplast suspensions and staining them with 20 pL of 0.5% (w/v) Evans blue indicator. The pro- portion of surviving protoplasts was evaluated by scoring 2000 to 5000 protoplasts for the number of unstained protoplasts. In the enzyme assays protoplast protein extracts were free of significant amounts of reducing sugars, as shown from blanks containing no added substrate. Protein concentration was determined using the Bradford method (1976) or with Coomassie Plus reagent.

RESULTS

ldentification of Two lsolated Acidic Tetrasaccharides

The water-soluble extract from semi-retted flax fibers was fractionated by size-exclusion chromatography on a polyacrylamide column (Fig. 1) that resulted in the resolu- tion of two tetrasaccharides, 1 and 2. Neutra1 sugar analysis showed that compounds 1 and 2 contained only rhamnose as a neutra1 sugar. The presence of a conjugated, unsaturated residue was strongly suggested by a maximum absor- bance at 230 to 235 nm (Davis et al., 1986a). Unsaturated GalpA residues are easily degraded during reduction or methylation reactions, and 4-linked GalpA residues are not fully carboxyl reduced. Also, base-catalyzed p-elimination sometimes leads to further degradation products. For these reasons, the structural analysis of compounds 1 and 2 was carried out by 'H- and 13C-NMR experiments and confirmed by FABMS.

The proton spectrum of compound 1 (Table I) showed three doublets between 1.20 and 1.32 ppm, easily assigned to the H-6,6,6 of rhamnose units (6 1.26: H-6 Rhacu, 1.28: H-6 Rhap, of a terminal reducing rhamnose, and 1.22 ppm: H-6 of an internal rhamnose unit).

In the region 4.6 to 5.9 ppm, eight doublets integrating as six protons were assigned to: (a) H-1a and H-1P of a reducing rhamnosyl residue at 6 5.21 and 4.91 ppm (3J1, , -1.7 and 1.1 Hz); (b) H-1 of a galacturonic acid unit directly linked to the rhamnose moiety located at the reducing end ( 8 5.17 and 5.06 ppm, 'J1, , -3.8 Hz); (c) H-la of an

3 2 1

Elution Volume Figure 1. Size-exclusion chromatography of a water-soluble extract from semi-retted flax fiber bundles on a Bio-Gel P-2 column.




Table 1. Chemical shifts (in ppm) of the proton resonances of compounds 1 and 2 'H ACalpA Rha GalpAa CalpAP Rhaa Rhap

H-1 5.1 1 (5.12)a

H-2 3.80 (3.83)

H-3 4.32 (4.33)

H-4 5.83 (5.99)

H-5

H-6

5.32 (5.14) 4.29

(4.29) 3.83

(3.82) 3.35

(3.37) 3.73

(3.66) 1.22

(1.22)

5.06 (5.26) 3.89

(4.1 3) 4.1 O

(5.35) 4.45 (4.58) 4.72 (4.99)

5.1 7 (5.1 5) 3.96

(4.1 6) 4.1 2

(5.31) 4.45

(4.58) 4.72 (4.99)

5.21 (5.21) 3.94

(3.98) 3.88

(3.89) 3.45

(3.50) 3.84

(3.86) 1.26

(1.27)

4.91 (4.92) 4.03 (4.06) 3.64

(3.80) 3.32

(3.35) 3.39

(3.41) 1.28

(1.29)

a Data in parentheses correspond to compound 2.

internal rhamnose (6 5.32 ppm); (d) H-5 of a galacturonic acid residue at S 4.72 ppm (Davis et al., 1986b); and (e) H-1 and H-4 of a 4,5-unsaturated galacturonic acid unit at 5.11 and 5.83 ppm (Mutter et al., 1996). These data showed that the anomeric protons integrated as four protons and strongly suggest that the structure of compound 1 is more than likely AGalA+Rha-+GalA-Rha.

The complete assignment of the proton spectrum was achieved by carrying out 2D-NMR COSY experiments and TOCSY 1D experiments. A11 proton data are reported in Table I.

The interglycosidic sugar linkages in the tetrasaccharide were then determined by doing 'H nuclear Overhauser effect difference experiments with selective saturations (Fig. 2). Saturation of H-1 of internal rhamnose at 5.32 ppm (Fig. 2B) gave a response on H-2 (4.29 ppm) of internal rhamnose and H-4 (4.45 ppm) of internal galacturonic acid. Thus, it showed that internal rhamnose is a linked through 0-4 to the internal GalpA unit. Saturation of H-1 of AGalpA at 5.11 ppm (Fig. 2C), with a response on H-2 AGalpA (3.8 ppm) and on H-2 of the internal rhamnose (4.29 ppm), showed that the 4,5-unsaturated galacturonic acid is linked through 0-2 to the internal rhamnose. Finally, saturation of H-1 GalpAa at 5.06 ppm of the internal galacturonic acid (Fig. 2D), with a response on H-2 GalpAa and H-2 of terminal reducing rhamnose (3.94 ppm), confirmed that the internal GalpA unit is a linked through 0-2 to the terminal reducing rhamnose residue. All of the experiments outlined above prove conclusively that the primary structure of compound 1 is a-~-AGalpA(1-+2)-a-~-Rhap (1-+4)-a-~- GalpA(1+2)-a,P-~-Rhap (Fig. 3).

The 'H-NMR spectrum of compound 2 is very similar to that of compound 1 except for the presence of a signal at 6 2.15 ppm, which integrated as three protons and is charac- teristic of an acetyl group. Assignment of the 'H-NMR spectrum was performed by doing a 2D-COSY experiment. The location of the acetyl group was easily determined through a comparison with the proton spectrum of compound 1. Data reported in Table I show that the signals that were most affected by this acetyl group occurred at S 5.35 and 5.31 ppm (downfield shift of 1.25-1.19 ppm) and corresponded, respectively, to H-3a and H-3P of the galacturonic acid residue. This indicated that the acetyl group is

located at the 0-3 position of the internal galacturonic acid. The primary structure of compound 2 was identified as a-~-AGalpA(1-+2)-a-~-Rhap(l~)-a-~-(3-O-ace~l)-GalpA (1+2)-a,P-~-Rhap (Fig. 3).

By using I3C/ 'H shift-correlated 2D experiments (Fig. 4), the l3C-NMR spectrum of compounds 1 and 2 were entirely assigned. The corresponding chemical shifts are reported in Table I1 and corroborate their primary structures given in Figure 3. The I3C-NMR spectrum of compound 2

H-2 R h a ~ n-2 G+AU

H-2 Rha

H-4 GalpA

, I H-2 Rha

I

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 PPM

Figure 2. 300-MHz nuclear Overhauser effect 'H experiments performed on tetrasaccharide 1 . A, 1 D 'H spectrum; B, nuclear Over- hauser effect difference with presaturation of H-1 of internal rhamnose unit at 5.32 ppm; C, nuclear Overhauser effect difference with presaturation of proton H-1 of the 4,5-unsaturated galacturonic acid residue at 5.1 1 ppm; and D, nuclear Overhauser effect difference with presaturation of proton H-1 of internal galacturonic acid at 5.06 ppm.




R = H : compound 1 R = Ac : compound 2

Figure 3. Structures of the two rhamnogalacturonide tetrasaccharides 1 and 2.

was entirely assigned as already described for compound 1, and the data are reported in Table 11.

The FABMS spectrum of sample 1, performed in the negative mode, showed an important molecular ion (M- H)- at mlz 643; this signal is consistent with the presence of the unsaturated tetrasaccharide. Loss of a water molecule gave the ion (M-H-H,O)- at mlz 625. Because of rupture of the glycosidic linkages the ions at mlz 485 and 339 and at 497 and 321 were observed. Other ions were formed that resulted from the cleavage of a ring: at mlz 539 and 363 and at mlz 527 and 381. Most of these fragment ions had a peak at mlz -18 with lower intensity, which corresponded to the loss of a water molecule. The FABMS spectrum of compound 2 showed a molecular ion at mlz 685. The main features of thr spectrum were peaks at mlz 539, 527, 381, and 321, which corresponded to fragmentation of the glycosidic bonds. With the loss of an acetyl group, three of these peaks gave an ion at mlz 497, 485, and 339. The FABMS results confirmed the chemical structures already given (Fig. 3) for compounds 1 and 2.

Biological Activities of the Two Rhamnogalacturonide Tetrasaccharides

R. fruticosus protoplasts or cells were incubated for O to 180 min in the presence of either the acetylated (compound 2) or unacetylated (compound 1) rhamnogalacturonide tetrasaccharide at a concentration between O and 3 p ~ . The viability of the protoplasts (or cells) during this treatment was verified and found to be as high as in control protoplasts (90%) or cells (95%).

When applied to suspended protoplasts for 15 min at 30 nM, compound 2 was observed to elevate the endogenous D-glycanases (1+3)-P-~-glucanase, pOly-(l+4)-P-D-ghICOS- aminidase, and (l-.4)-a-~-galacturonase with R values of about 17, 12, and 4, respectively, but it did not promote D-glycosidase (P-D-xylosidase) activation (Fig. 5A, bars 2). In marked contrast, compound 1 attenuated the laminarinase activation ( R = ll), did not elicit chitinase or (1+4)- a-D-galacturonase, but enhanced the P-D-xylosidase response ( R = 6.7) (Fig. 5A, bars 1). After 4 to 5 h of treatment, each of the short-term reported activities (except that of P-D-xylosidase) increased significantly. For in- stance, after 36 h compound 2 gave laminarinase and chitinase activation with R values of 45 and 32, respectively.

Cell-suspension cukures exposed to 30 nM of inducer 1 or 2 for 15 min yielded the following responses: laminarinase and chitinase activations by compound 2 were some-

what reduced, with R values of 7 and 4, respectively, and the (1+4)-a-~-galacturonase or P-D-xylosidase activity could not be detected (Fig. 5B, bars 2). Compound 1 also promoted attenuated responses in cells, since laminarinase and p-D-xylosidase activation exhibited R values of 5 and 4, respectively (Fig. 5B, bars 1).

Kinetic studies of the P-D-xylosidase activation in protoplast suspensions (Fig. 6A, curve a) showed that compound 1 at 25 nM rapidly initiated a cyclic pattern of transient increases in P-D-xylosidase activity, with successive activity peaks of lower amplitude but broader bases. The induced response peaked after 10 min ( R = 5.7), 90 min ( R = 4.2), and 180 min ( R = 1.8). The influence of the sugar concentration on the activation of P-D-xylosidase revealed a bell-shaped dose-response curve (Fig. 6B, curve a). The enzyme activation was maximal ( R = 5.7) at an effector concentration of about 25 nM, whereas no response was induced by concentrations lower than 0.15 nM or higher than 3 p ~ . In suspended cells kinetic curves (Fig. 6A, curve b) indicated that P-D-xylosidase activation also followed a bell-shaped curve, but the detected response was both markedly delayed and attenuated ( R = 3 at 30 min; R = 2.2 at 90 min; and R = 1.1 at 180 min). Furthermore, as shown in Figure 6B, curve b, the treatment of cells for 10 min by

6.0 5.5 5.0 4.5 4.0 3.5 310PPM

Figure 4. Contour plot of 2D heteronuclear H/13C shift-correlated spectrum at 300175 MHz of tetrasaccharide 1 in D,O.




Table II. Chemical shifts (in ppm) of the 13C resonances of compounds I and 2 ' 3c ACalpA Rha CalpAa CalpAp Rhaa Rhap

c-1 99.76 100.38 99.65 102.79 93.42 95.58

c-2 71.90 78.96 69.80 70.49 78,85 81.99 (71.63) (79.85) (68.63) (68.02) (79.31) (81 34 )

c-3 67.68 71.31 71.97 72.10 71.1 6 73.84 (67.38) (71.42) (74.21) (74.21) (70.38) (71.42)

c-4 109.65 73.77 79.05 79.05 73.97 73.75 (1 11.40) (73.82) (76.93) (76.49) (73.69) (71.23)

c-5 146.51 70.77 73.1 1 72.88 70.35 74.00 (1 42.6) (71.1 5) (72.71) (72.71) (71.1 5) (73.91)

C-6 170.29 18.42 175.70 175.70 18.42 18.55 (173.19) (1 8.33) (1 74.64) (1 74.71) (1 8.50) (18.71)

(1 01.02)= (1 00.25) (99.7 3) (1 02.48) (93.44) (95.55)

a Data in parentheses correspond to compound 2.

compound 1 resulted in a plateau response curve raising the maximal R value of 3. It is worth noting that in the presence of transcription inhibitor (1 pM.cycloheximide) or of translation inhibitor (1 pg mL-' actinomycin D) the short-term responses up to 180 min were fully maintained.

Kinetics of the laminarinase activation in protoplasts induced by compound 2 used at 30 nM could exhibit a pattern similar to the cyclic pattern of the induction of P-D-xylosidase by compound 1, since the oscillation of the early detected response peaked after 10 to 20 min ( R value of about 23) and at 120 min ( R value of about 15). Incuba- tion for a longer duration (up to 36 h) resulted in a very large increase of response, since an R value of 45 was detected. In the presence of an inhibitor of transcription or translation, the short-term laminarinase response was fully maintained, whereas the long-term treatment resulted in a markedly attenuated response (40-50% inhibition at 36 h).

Compounds 1 and 2 acting for 30 min at 30 nM were also found to promote the induction of PI in R. fruticosus protoplasts. By reference to PI prepared from controls (nontreated protoplasts), only the nonacetylated compound 1 induced a very efficient PI. This PI equivalent to 0.5 pg of protein gave inhibition of trypsin activity up to 15% control (trypsin activity without effector), whereas the PI prepared from protoplasts incubated with compound 2 or from controls gave inhibition values of only 4 to 6%. The ability of the PI induced by compound 1 to inhibit trypsin activity was found to linearly increase with PI quantities up to 2 pg of protein. To assess whether PI could change D-

glycohydrolase activity in vitro, enzyme extracts isolated from protoplasts treated by inducer 1 and 2 and from controls were incubated in the presence of an effector (0.5 pg of protein). It was noticed that only (1+4)-a-~- galacturonase activity increased in the presence of a PI, and that only the PI induced by compound 1 was active. As shown in Figure 7, bar lb, it was efficient on LY-D-

A

25 -1

Laminarinase I

O E 9 15

a-l)-galacturonase j 10

N 5 6 O

B

Laminarinase

2

P-Q-xylosidase

a-i)-galacturonase

5 . (1 +3)-P-~-Clucanase (laminarinase), poly-(l+4)-P-D- " galacturonase from protoplasts treated by compound 1, since its action resulted in a marked increase of enzyme response with an R Vahe of 10. It was less active (Fig. 7, bar 2b) or even inactive (Fig. 7~ bar Tb) On enzyme isolated

Figure glucosaminidase (chitinase), P-o-xylosidase, and (1-4)-a-D- galacturonase (D-galacturonase) activation in R. fruticosus protoplasts (A) or cells (6) challenged by compound 1 or 2. 2 X 1 O6 Drotoalasts (cells) in 25 mL of buffer were incubated for 15 min in the . . , .

from protoplasts treated bY ComPound 2 Or from controls. It was noticed that neither the short-term induction of PI nor PI properties were changed in the presence of a transcription or translation inhibitor.

presence of 30 n M sugar. Each plot was obtained by least-squares regression of data from four replications carried out from two independent elicitation sets. R is reported as the rate of D-glycohydrolase activity in treated protoplasts (cells) over controls.




A

O 50 100 150 200 250

T”>

B

r i 3 v1 d

i 2 1

I /

-1 O 1 2 3 4 COMPOUND 1 LOG (C, nM)

Figure 6. Time course (A) and dose response (B) for fl-o-xylosidase activation by compound 1 in R. fruticosus protoplasts (curve a) or cells (curve b). In A, 2 X 10‘ protoplasts (cells) in 25 mL of buffer were incubated for O to 240 min in the presence of 25 nM sugar. In B, protoplasts (cells) were treated for 15 min by sugar u p to a 3 p~ concentration. Each curve was obtained by least-squares regression of data from 8 to 1 O replications carried o u t from four independent elicitation sets. R is reported as the rate of P-o-xylosidase activity in treated protoplasts over controls.

DI SC USSl ON

The main compounds isolated from water extraction of semi-retted flax fiber bundles were a mixture of two tetrasaccharides. They were shown to possess a rhamnosyl residue at the terminal reducing end and a 4,5-unsaturated GalpA residue at the terminal nonreducing end. One of the tetrasaccharides had an acetyl group at the 0-3 position of interna1 galacturonic acid residue, as already described by Komalavislas and Mort (1989). It is obvious from the production of these two unsaturated tetrasaccharides that lyase was involved in the retting process. P-Elimination lyase cleavage was deduced to have occurred between the

Rha and the GalA units of pectin polysaccharides, with an alternating sequence of RG-I type. Such action implies that the lyase was a rhamnogalacturonan-a-L-rhamnopyranosyl- (1+4)-a-~-galactopyranosyl uronide lyase, as already described from AspergiZlus aculeatus by Mutter et al. (1996). The rhamnosyl residues were not ramified, either because they were not ramified in the original pectins or because arabinases and galactanases produced during the retting process have cleaved the arabínogalactan side chains.

Previously, severa1 oligosaccharides with alternating rhamnose and galacturonic acid units have been isolated. For example, a-~-hGalpA( 1-+2)-a,P-~-Rhap has been obtained from a mucilage of germinated cress seeds (Hasegawa et al., 1992), and ~u-~-GalpA(1+2)-a-~-Rhap and (3-0- acetyl)a-D-GalpA(l+Z)-L-Rhap disaccharides have been isolated from carrot, cotton, tobacco, and tomato cell walls (Komalavislas and Mort, 1989). Homologous oligomers (degree of polymerization 6-20) with a repetitive structure based on the -4)-a-~-GalpA(1-+2)-~-Rhap(l+ unit were isolated from apple, beet, and citrus pectins (Renard et al., 1995). Furthermore, four oligosaccharides with the alter- nated structure @-~-Rhap(l-+4)-a-~-GalpA(l-+2)-P-~- Rhap(l+4)-~-GalpA, bearing zero, one, or two Gal residues linked to the 0-4 of the rhamnose units, were obtained after rhamnogalacturonase hydrolysis of apple pectins (Colquhoun et al., 1990; Schols et al., 1990). These last oligomers differ from the tetrasaccharides obtained during the work outlined in this paper, because their reducing end is a GalpA unit and they lack a AGalpA unit. Recently,

1

b

0 4 Figure 7. In vitro modulation of (1 ~4)-a-~-gaIacturonase in R. fruticosus protoplasts by PI. A total of 2 X 1 O6 protoplasts in 25 mL of buffer was incubated for 30 min in the presence of compound 1 or 2 (25 nM), and (l-t4)-c~-D-galactUronase and PI were extracted; T indicates the controls. In vitro a-o-galacturonase was quantified in absence of PI (o) or in presence of PI (0.5 pg of protein) obtained from the controls (a) or from the protoplasts treated by compound 1 (b) or 2 (c). Each plot was obtained by least-squares regression of data from six replications carried out from three independent elicitation sets. R is reported as the rate of a-D-galacturonase activity in treated protoplasts (or in controls in presence of PI) over controls without PI.




Mutter et al. (1996), by action of a rhamnogalacturonan lyase on a RG-I, reported a hexasaccharide with the following structure:

AGalpA(1 + 2)-p-~-Rhap(l + 4)-a-~-GalpA(1 2)-~-Rhap 4 4

1 1 t t

p-~-Galp P-D-Galp

It has been demonstrated here that the two rhamnogalacturonide tetrasaccharides are signaling molecules in R. fruticosus protoplasts (or cells). One of these sugars, compound 1, was found to rapidly promote, in less than 30 min, changes in the activity of enzymes or proteins, which are biochemical indicators of defense metabolism in higher plants. They increased the activities of the enzymes laminarinase and chitinase, and of the Ser proteinase inhibitor. They also induced P-D-xylosidase and (1 -+4)-a-~-galacturonase activities, which are able to hydrolyze plant or pathogen cell walls. In different bioassays concerning sev- era1 aspects of plant defense or plant development, the optimal degree of polymerization for active-signaling homogalacturonan oligosaccharides is often about 10 to 12 (Darvill et al., 1992). As described here, it was observed that a tetrameric rhamnogalacturonan sequence retained biological activity and that the acetyl group at the 0-3- position of the interna1 galacturonide residue had a particularly important biological function since it induced specific enzymes. Until now, the bioactivity of heteropectins has been reported only twice: from a rhamnogalactan polymer isolated from sycamore callus and described as an elicitor of phytoalexíns (Ryan et al., 1981), and more recently from the dimeric structure a-~-AGalpA-(l+2)-a, p-L-Rhap secreted by germinated cress seeds that either promoted hypocotyl growth or inhibited root growth (Ha- segawa et al., 1992).

The flax rhamnogalacturonides that have been reported in this paper were bioeffective at nanomolar concentrations with an optimal concentration of approximately 25 nM. Also, induced enzyme activation did not require the pres- ente of the cell wall. Furthermore, the effector responses in protoplasts were very rapid and transient, suggesting their involvement in the early signal transduction cascade. These findings indicated that the tetramers can act as a signal perceived at the plasma membrane level. Other signaling molecules tested in the laboratory also triggered transient and repetitive responses. The responses were shown to be mediated by an energy- and temperature-dependent process, since they were blocked when l mM KCN was added in protoplast suspensions and they were largely inhibited at 4°C (Y. Liénart, unpublished results). lZ5I-labeled polygalacturonic acid elicitor was found to be first associated at the cell surface, and then internalized via a receptor- mediated endocytotic pathway (Horn et al., 1989). Detec- tion of oligosaccharides by protoplasts has been reported in a number of plant systems (Dangl et al., 1987; Kauss et al., 1989; Liénart et al., 1991). Signal recognition is thought to involve membrane receptors (Cheong et al., 1993; Shibuya et al., 1993) and changes in membrane depolarization (Ku- chitsu et al., 1993). Concerning the mechanism of oligoga-

lacturonide activity, lower concentrations of size-specific oligomers are known to induce, within 5 min, a transient stimulation of K+ efflux, Ca2+ influx, cytosolic acidification (Mathieu et al., 1991), and oxidative response (Davies et al., 1993). In agreement with the idea that they are anti-auxins (Branca et al., 1988; LoSchiavo et al., 1991), the oligogalacturonides were also found to interact with auxin-binding membrane sites (Filippini et al., 1992).

Even though the perception mechanism of the tetramers in R. fruticosus has not been conclusively determined, a maximal signaling effect had been observed within 15 min, and the short-term induced responses were fully maintained in the presence of an inhibitor of transcription or translation. The findings suggest that the early detected responses result in posttranslational modification of preexisting stored proteins, rather than in a mediation by synthesis of mRNA and of protein de novo. Also, it is worth noting that the binding of compounds 1 or 2 to receptors may be essential for invoking the short-term reported responses.

ACKNOWLEDCMENTS

We thank S. Lafond for biological experiments, and C. Gey and ' C. Bosso for measuring the NMR and FABMS spectra, respectively.

Received March 24, 1997; accepted July 14, 1997. Copyright Clearance Center: 0032-0889 / 97 / 115 / 0793 / 09.

LITERATURE CITED

Aposto1 I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells. Role in defense and signal transduction. Plant Physiol 90: 109-1 16

Bradford HM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Ana1 Biochem 72: 248-254

Branca C, D e Lorenzo G, Cervone F (1988) Competitive inhibition of the auxin-induced elongation by a-D-oligogalacturonides in pea stem segments. Physiol Plant 72: 499-504

Broeckaert WF, Peumans WJ (1988) Pectic polysaccharides elicit chitinase accumulation in tobacco. Physiol Plant 7 4 740-744

Bruce RJ, West CA (1989) Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. Plant Physiol91: 889-897

Campbell AD, Labavitch JM (1991) Induction and regulation of ethylene biosynthesis and ripening by pectic oligomers in tomato pericarp discs. Plant Physiol 97: 706-713

Cheong J-J, Alba R, Côté F, Enkerli J, Hahn MG (1993) Solubili- zation of functional plasma membrane-localized hepta /3- glucoside elicitor-binding protein from soybean. Plant Physiol

Colquhoun IJ, de Ruiter GA, Schols HA, Voragen AGJ (1990) Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase. Carbohydr Res 206: 131-144

Dangl JL, Hauffe KD, Lipphardt S, Hahlbrock K, Scheel D (1987) Parsley protoplasts retain differential responsiveness to U.V. light and funga1 elicitor. EMBO J 6: 2551-2556

Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors: a defense against microbial infection in plants. Annu Rev Plant Physiol35: 243-275

Darvill AG, Augur C, Bergmann C, Carlson RW, Cheong J-J, Eberhard S, Hahn MG, Lo V-M, Marfi V, Meyer B, and others (1992) Oligosaccharins: oligosaccharides that regulate growth, development and defense responses in plants. Glycobiology 2:

Davies D, Merida J, Legendre L, Low PS, Heinstein P (1993) Independent elicitation of the oxidative burst and phytoalaexin formation in cultured plant cells. Phytochemistry 32: 607-611

103: 1173-1182

181-198




Davis EA, Derouet C, Herve du Penhoat C (1990) Isolation and N.M.R. study of pectins from flax (Linum usitatissimum L.). Car- bohydr Res 197: 205-215

Davis KR, Darvill AG, Albersheim P, De11 A (1986a) Host- pathogen interactions. XXIX. Oligogalacturonides released from sodium polypectate by endopolygalacturonic acid lyase are elicitors of phytoalexins in soybean. Plant Physiol80 568-577

Davis KR, Darvill AG, Albersheim P, De11 A (1986b) Host- pathogen interactions. XXX. Characterization of elicitor phytoalexin accumulation in soybean released from soybean cell-walls by endopolygalacturonic acid lyase. 2 Naturforsch 41c: 3948

Davis KR, Hahlbrock K (1987) Induction of defense responses in cultured parsley cells by plant cell-wall fragments. Pla& Physiol 8 5 1286-1290

Filippini F, LoSchiavo F, Terzi M, Branca C, Bellincampi D, Salvi G, Desiderio G, De Lorenzo G, Cervone F (1992) Phytoalexin elicitor-active a-1.4-u-oligolacturonides reduce auxin perception by plant cells and tissues. ln CM Carsen, LC Van Loon, D Vreugdenhil, eds, Progress in Plant Growth Regulation. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 517-521

Guillon F, Thibault JF, Rombouts FM, Voragen AGJ, Pilnik W (1989) Enzymatic hydrolysis of the "hairy" fragments of sugar- beet pectins. Carbohydr Res 190: 97-108

Hasegawa K, Mizutani J, Kosemura S , Yamamura S (1992) Isola- tion and identification of lepidimoide, a new allelopathic sub- stance from mucilage of germinated cress seeds. Plant Physiol

Horn MA, Heinstein PF, Low PS (1989) Receptor-mediated endo- cytosis in plant cells. Plant Cell 1: 1003-1009

Hustache G, Mollard A, Barnoud F (1975) Culture illimitée d'une souche anergiée de Rosa gfauca par la technique des suspensions cellulaires. C R Acad Sci Paris 281D: 1381-1384

Kauss H, Jeblick W, Domard A (1989) The degrees of polymerization and the acetylation of chitosan determine its ability to elicit callose formation in suspended cells and protoplasts of Catharanthus roseus. Planta 178: 385-392

Komalavilas P, Mort AJ (1989) The acetylation at 0-3 of galacturonic acid at the rhamnose rich portion of pectins. Carbohydr Res 189: 261-272

Kuchitsu K, Kikuyama M, Shibuya N (1993) N-Acetylchitooligo- saccharides, biotic elicitor for phytoalexin production, induce transient membrane depolarization in suspension-cultured rice cells. Protoplasma 174: 78-87

Lau JM, McNeil M, Darvill AG, Albersheim P (1985) Structure of the backbone of rhamnogalacturonan I, a pectic polysaccharide in the primary cell-walls of plants. Carbohydr Res 137: 111-125

Lee YE, Zeikus JG (1993) Genetic organization, sequence and biochemical characterization of recombinant P-xylosidase from Thermoanerobacterium saccharolyticum strain B6A-RI. J Gen Micro- biol 139: 1235-1243

Liénart Y, Gautier C, Driguez H (1991) Silica-boundN-acetylglucos- aminyl residues elicit laminarinase activity in Rubus protoplasts. Plant Sci 77: 41-45

LoSchiavo F, Filippini F, Cozzani F, Vallone D, Terzi M (1991) Modulation of auxin-binding proteins in cell suspensions. I. Differential responses of carrot embryo cultures. Plant Physiol 97: 60-64

Massiot P, Rouau X, Thibault JF (1988) Characterisation of the extractable pectins and hemicelluloses of the cell-wall of carrot. Carbohydr Res 172 229-242

Mathieu Y, Kurkdijan A, Xia H, Guern J, Koller A, Spiro M, ONeill M, Albersheim P, Darvill AG (1991) Membrane responses induced by oligogalacturonides in suspension-cultured tobacco cells. Plant J 1: 333-343

McNeil M, Darvill AG, Albersheim P (1980) Structure of plant cell-walls. X. Rhamnogalacturonan I, a structurally complex pectic polysaccharide in the walls of suspension-cultured sycamore cells. Plant Physiol 66: 1128-1134

Melotto E, Greve LC, Labavitch JM (1994) Cell-wall metabolism in ripening fruit. VIL Biologically active pectin oligomers in

100 1059-1061

ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant Physiol 106: 575-581

Morvan C, Jauneau A, Voreux H, Morvan O, Demarty M (1990) Contribution de5 ciments aux caractéristiques physicochimiques des fibres de lin: application B un rouissage enzymatique. C R Soc Biol 184 18-30

Morvan O, Jauneau A, Morvan C, Voreux H, Demarty M (1989) Biosynthèse des pectines et différentiation des fibres cellulo- siques au cours de la croissance du lin. Can J Bot 67: 135-139

Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan a-~-rhamnopyranosyl-(l+4)-cu-~-galacto- pyranosyluronide lyase. Plant Physiol 110: 73-77

Redgwell RJ, Selvendran RR (1986) Structural features of cell- wall polysaccharides of onion Allium cepa. Carbohydr Res 157

Renard CMGC, Crépeau M-J, Thibault JF (1995) Structure of the repeating units in the rhamnogalacturonic backbone of apple, beet and citrus pectins. Carbohydr Res 275: 155-165

Renard CMGC, Voragen AGJ, Thibault JF, Pilnik W (1991) Stud- ies on apple protopectin. V. Structural studies on enzymatically extracted pectins. Carbohydr Polym 1 6 137-154

Rickauer MJ, Fournier J, Esquerré-TugayC MT (1989) Induction of proteinase inhibitors in tobacco cell suspension culture by elicitors of Phytophtkora parasitica var Nicotiana. Plant Physiol 9:

Robertsen B (1986) Elicitors of the production of lignin-like compounds in cucumber hypocotyls. Physiol Mo1 Plant Pathol 28:

Ryan CA, Bishop P, Pearce G, Darvill AG, McNeil M, Alberheim P (1981) A sycamore cell-wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor inducing activities. Plant Physiol 68: 616-618

Saulnier L, Brillouet JM, Joseleau JP (1988) Structural studies of pectic substances from the pulp of grape berries. Carbohydr Res 182: 63-78

Schols HA, Posthumus MA, Voragen AGJ (1990) Structural features o€ hairy regions o€ pectins isolated from apple juice pra- duced by the liquefaction process. Carbohydr Res 206 117-129

Schols HA, Verhuis E, Bakx EJ, Voragen AGJ (1995) Different populations of pectic hairy regions occur in apple cell-walls. Carbohydr Res 275: 343-360

Shibuya N, Kaku H, Kuchitsu K, Maliarik MJ (1993) Identifica- tion of a nove1 high-affinity binding site for N-acetylchitooligo- saccharide elicitor in the microsomal membrane fraction from suspension-cultured rice cells. FEBS Lett 329 75-78

Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:

Spiro MD, Kates KA, Koller AL, ONeill MA, Albersheim P, Darvill AG (1993) Purification and characterization of biologically active 1,4-linked a-u-oligogalacturonides after partia1 di- gestion of polygalacturonic acid with endopolygalacturonase. Carbohydr Res 247: 9-20

Stevens B JH, Selvendran RR (1984) Structural features of cell-wall polymers of the apple. Carbohydr Res 135: 155-166

Thibault JF, Renard CMGC, Axelos MAV, Roger P, Crepeau MJ (1993) Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr Res 238 271-286

Thomas JR, Darvill AG, Albersheim P (1989) Isolation and structural characterization of the pectic polysaccharide rhamnogalacturonan I1 from walls of suspension-cultured rice cells. Carbo- hydr Res 185: 261-277

Tran Thanh Van K, Toubart P, Cousson A, Darvill AG, Gollin DJ, Chelf P, Albersheim P (1985) Manipulation of the morpho- genetic pathway of tobacco explants by oligosaccharins. Nature

Vignon MR, Garcia-Jaldon C (1996) Structural features of the pectic polysaccharides isolated from retted hemp bast fibers (Cannabis sativa). Carbohydr Res 296: 249-260

183-199

1065-1070

137-148

19-23

314: 615-617



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