Enhancement of growth by expression of poplar ... - Wiley Online Library

The Plant Journal (2003) 33, 1099±1106

Enhancement of growth by expression of poplar cellulase in Arabidopsis thaliana Yong Woo Park1, Rumi Tominaga1, Junji Sugiyama1, Yuzo Furuta2, Eiichi Tanimoto3, Masahiro Samejima4, Fukumi Sakai1 and Takahisa Hayashi1, 1 Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, 2 Biological Function Science Course, Kyoto Prefectural University, Kyoto 606-8522, Japan, 3 Department of Information and Biological Sciences, Nagoya City University, Nagoya 467-8501, Japan, and 4 Department of Biomaterial Sciences, University of Tokyo, Tokyo 113-8657, Japan Received 19 October 2002; accepted 6 December 2002. For correspondence (fax 81 774 38 3600; e-mail [email protected]).

Summary To study the role of cellulose and cellulase in plant growth, we expressed poplar cellulase (PaPopCel1) constitutively in Arabidopsis thaliana. Expression increased the size of the rosettes due to increased cell size. The change in growth was accompanied by changes in biomechanical properties due to cell wall structure indicative of decrease in xyloglucan cross-linked with cellulose micro®brils by chemical analysis and nuclear magnetic resonance (NMR) spectra. The result supports the concept that the paracrystalline sites of cellulose micro®brils are attacked by poplar cellulase to loosen xyloglucan intercalation and this irreversible wall modi®cation promotes the enlargement of plant cells. Keywords: poplar cellulase, mechanical properties, leaf growth, Arabidopsis, xyloglucan.

Introduction Growth in plant cells depends in part on the mechanical properties of the cell wall, where a complex material comprising ®brous cellulose micro®brils is embedded in an amorphous matrix of polysaccharide and protein. While the structures of the individual components have been reasonably well elucidated for some time, less is known about the process in which the components assemble into a functional structure and how the characteristics of that assembly govern expansion together. Cellulose is formed as micro®brils which form the skeleton in higher plants. As cellulose micro®brils are fundamental for the cell wall, these provide mechanical strength and control expansion. Although cellulose synthesis is reasonably well understood as is the structure of an ideal micro®bril, we know much less about the structure of the micro®bril within the wall. The micro®brils have 1,4-b-glucan chains, not only in, each glucose unit that is upside down with respect to the preceding one with intramolecular hydrogen bonds between O-50 and O-30 (distance 0.26 nm) as well as between O-20 and O-60 (distance 0.36 nm), and also have 1,4-b-glucan chains in intermolecular hydrogen bonds which hold about 30±200 chains ß 2003 Blackwell Publishing Ltd

together for making micro®brils. The cellulose framework also embeds xyloglucans and proteins (enzymes) to form a cell wall network, where xyloglucans may bind nascent 1,4b-glucan and intercalate to form paracrystalline micro®brils (Hayashi, 1989). This site has previously been studied by looking at xyloglucan endotransglycosylase or expansin, but the results are not always clear. Our approach has been to overexpress a plant cellulase because the cellulase can attack certain structural motifs of the cell wall. The question is whether plant cellulases could generate the loosening of load-bearing xyloglucan tether between micro®brils by hydrolysis of the 1,4-b-glucans. In part I of this study, the function of poplar cellulase has been examined using sense and antisense poplar transgenic plants (trg), but poplar cellulase was repressed even in sense transformants of poplar (Ohmiya et al., 2002). Therefore, poplar cellulase cDNA including the signal sequence was fused with a constitutive promoter with enhancer and the effect of the gene was tested in Arabidopsis thaliana. The question is whether the overexpression of poplar cellulase promotes the expansion growth of leaf in A. thaliana by the endohydrolysis of 1,4-b-glucan for cellulose deposition. 1099

1100 Yong Woo Park et al. Plant cellulases (EC 3.2.1.4) that catalyze the cleavage of the internal 1,4-b-linkages of cellulose have been proposed to control various aspects of plant development, such as abscission (Lewis and Koehler, 1979), fruit softening (Fischer and Bennett, 1991), wall loosening (Hayashi et al., 1984), vascular differentiation (Sheldrake, 1970) and symbiosis (Verma et al., 1978). Various functions have been proposed for these hormone-induced enzymes in plant growth, all of which could involve their potential participation in the metabolism of cellulose micro®brils. The aim of this study was to assess the action of poplar cellulases on the formation of cellulose micro®brils in the model plant overexpressing cellulase. The impact of the paper is to reveal the basic cellulose framework of linkages between cellulose and xyloglucan in the primary wall.

Results Expression of PopCel1 To study the effect of cellulase on growth and cell wall structure, we generated transgenic Arabidopsis plants that expressed a cellulase from poplar (PaPopCel1) under the control of a constitutive promoter. To assay the expression of the transgene, we used an antibody against a 15-amino acid sequence (163CWERPEDMDTPRNVY167) common to both PopCel1 and PopCel2. In a transgenic line (trg1), the antibody recognized a single, 50-kDa band on a Western blot, present in the root, stem, and leaf (Figure 1a), corresponding to the expected size of the poplar cellulase. The signal was found in the apoplastic and wall-bound fractions in the leaves of the transgenic plants, although a faint signal was also detected in their cytosol fraction. The level of expression was different among the transgenic plants: it was relatively high in trg1, but relatively low in trg2. Thus, the poplar cellulase was present in the cell walls. The activity of cellulase in the leaves harvested at 30 days after sowing varied among the transgenic plants (trg1±4) and was two- to ®vefold higher than that in the wild-type plant (wt) (Figure 1b). Activity of cellulase was also assessed by measuring soluble cello-oligosaccharides, which are presumably released by the enzyme. These oligosaccharides accumulated in the transgenic plants, and their amounts were closely related to cellulase activities among the four transgenic lines, but were increased greatly compared to the cellulase activity in the wild type. Increased cellulase in the cell wall might be expected to decrease the levels of cellulose, but in fact cellulose per plant increased to nearly the same extent as did cellulase. This increased cellulose re¯ects the larger size of the plants and cellulose per milligram of dry weight was not changed (see below).

Figure 1. Localization of PaPopCel1 gene product and the level of cellulase activity and cello-oligosaccharides in leaf. (a) Western blot of the gene product. A, apoplastic fraction; C, cytoplasmic fraction; W, wall-bound fraction. (b) Level of cellulase activity and cello-oligosaccharides in leaves. Three independent plants for each clone were used for the determination.

Growth of transgenic plants The transgenic plants produced rosettes that were about 1.6-fold larger in diameter than those of the wild type (60 wild-type plants) (Figure 2a). The overall morphology of rosettes in the transgenic plants was similar to that of the wild-type (Figure 2b). Leaf length and width were increased to the same extent as was the length of the blade and the petiole, and all leaves including the cotyledons were affected (Figure 2c). Additionally, the transgenic lines made more leaves than the wild type, probably because the higher growth rate shortened the plastochron. Parenchyma cells in the transgenic and wild-type leaves were identi®ed in the ®fth leaf (Tsuga et al., 1996) and both palisade and epidermal cells were larger in the leaves of the transgenic plant (trg1) than in the leaves of the wild-type plant (Figure 2d). When seedlings were grown on agar plates in the dark for 4 days, the transgenic plants had 1.14 (trg2)- to 1.16 (trg1)fold longer hypocotyls than the wild-type plant (Table 1). The morphology of dark-grown hypocotyls in the transgenic plants was also similar to that in the wild-type plant. The numbers of epidermal cells per stem in hypocotyls were identical in the transgenic and wild-type plants. We conclude that both epidermal and parenchyma cells in the hypocotyls, as well as in the leaves were larger in the transgenic plants than in the wild-type plant.

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 1099±1106

Overexpression of poplar cellulase

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Figure 3. Effect of PaPopCel1 transgenes on relative load±extension curves. The load versus extension curves are plotted as relative values of each leaf blade strip, where the breakage of the strip occurs. Five leaf blade strips for each clone were used for the determination and their averaged curves are shown.

Figure 4. Area-averaged spectra of 13C NMR for the 4% KOH-insoluble fraction from transgenic Arabidopsis leaves. Insert shows C6 region where substantial differences are seen among specimens.

Table 1 Length of etiolated hypocotyls

Figure 2. Effect of PaPopCel1 transgenes on rosette leaf and cell growth. (a) Histograms of rosette sizes in the transgenic (black bar) and wild-type (white bar) plants from 30-day-old plants. (b) Rosettes from 30-day-old T3 plants. Bar 4 cm. (c) Alignment of rosette leaves of the transgenic plants shown in (b), arranged left to right from ®rst to last leaves emerge. (d) Palisade (left) and epidermis (right) cells. Bar 100 mm.

Plant

Length (mm)

wt trg1 trg2 trg3 trg4

10.26 11.89 11.68 11.70 11.78

0.36 0.44 0.43 0.38 0.41

Twenty 4-day-old seedlings were used for the determination of hypocotyl length.


1102 Yong Woo Park et al. Table 2 Mechanical properties of leaf sections Plant

Breaking load (g)

Total extensibility at breaking point (mm)

Plastic extensibility (% dl)

Osmolality (kPa)


7.70 7.41 7.89 7.63 7.50

1.85 2.31 2.77 2.62 2.46

9.70 12.75 11.10 13.15 12.45

765 763 780 775 765

1.34 0.93 1.03 0.76 0.84

0.20 0.27 0.18 0.28 0.22

1.5 0.9 0.8 0.9 0.8

27 24 24 34 24

Twenty independent leaf blade strips which do not contain main vein were used for the determination of mechanical properties.

Mechanical properties of leaf cell walls

Structure of cell walls

To examine the cell wall extensibility by a creep test, strips were excised from the growing leaf, treated with methanol to kill cells and remove membranes, and allowed to creep under a constant load (5 g for 5 min) before release. The total extensibility at breaking point was higher in the transgenic plants than in the wild-type plant but no signi®cant difference was observed in the breaking load. The plastic extensibility at 5-g load was higher in the transgenic plants than in the wild-type plant, but no signi®cant difference was observed in the osmotic potential of the cell sap between the transgenic and wild-type leaves (Table 2). This is in agreement with the earlier ®nding (Yuan et al., 2001) that fungal endo-1,4-b-glucanase caused a marked increase in wall plasticity in the hypocotyl walls of cucumber and the coleoptile walls of wheat. To compare the mechanical properties of leaves further, we further analyzed load elongation with live leaf blade strips. Figure 3 shows the load±extension curves plotted as relative load against extension values of the breakage point, with each value of max-extension. The load and elongation values were expressed as relative values of breaking point for comparison among the transgenic and wild-type plants as the break-off points of leaves were different from each other and the cross-sections of samples could not be measured. The transgenic plants had larger elongation values than the wild-type plant (Figure 3 inset). The load±extension curves of leaf blades of the transgenic plants (trg1±4) showed an arch pattern above the curves for the wild-type plant, although the leaf blade is composed of randomly directed mesophyll cells and minor veins. Generally, the load±extension curves of brittle material with many cross-linkings are more linear and brittle materials elongate less. In addition, the brittle materials with many cross-linkings show load±extension curves with a shallower slope when the load and elongation values are expressed as relative values of breaking point. Therefore, the typical arch pattern and the large break point value of elongation show that the transgenic plants have fewer cross-linking components than the wild-type plant.

A macromolecular complex composed of xyloglucan and cellulose was prepared as a 4% KOH-insoluble fraction from the cell wall preparation of leaves in accordance with a previous report (Hayashi and Maclachlan, 1984). As shown in Table 3, the transgenic plants contained similar amounts of cellulose but retained less xyloglucan in the 4% KOHinsoluble fraction than the wild-type plant. The sum of the xyloglucan in soluble and insoluble fractions did not differ among the genotypes. The amounts of minor components in all the transgenic plants were similar to those in the wildtype plant. The methylated sugars due to the minor components consisted of 4-linked xylose, 2,4-linked xylose, and 4-linked galactose at the constant proportion in both the transgenic and wild-type plants. These methylated sugars are probably derived from xylan and galactan in a ratio of 7 : 3. Therefore, the transgenic plants differ from the wildtype plant only in the amount of xyloglucan present in the 4% KOH-insoluble fraction. Micro®bril structure was also analyzed with nuclear magnetic resonance (NMR). A conventional cross-polarization magic angle spinning (CP/MAS) technique was employed without any spectral editing (Larsson et al., 1997), and spectra were area-averaged in a certain region of interest to allow comparison. In cell wall preparations, the broad peak around 101 p.p.m. has been identi®ed as xyloglucan (Figure 4), which may be a typical C1 resonance of galactosyl, glucosyl, and xylosyl residues (Gidley et al., 1991; Table 3 Xyloglucan and cellulose contents (in mg mg 1 leaf dry weight) in the 4% KOH-soluble and -insoluble fractions of leaf Soluble fraction Insoluble fraction Plant Xyloglucan

Minor Xyloglucan Cellulose components


83 37 42 37 38

150 188 187 189 190

190 187 189 191 185

9 6 7 8 5


Overexpression of poplar cellulase Whitney et al., 1995). There was no difference in this signal from xyloglucan between transgenic and wild-type plants. A close investigation revealed that there were small but substantial differences between the transgenic and wildtype plants, especially at 89 and 84 p.p.m. for C4, and at 66 and 63 p.p.m. for C6, resonances indicative of cellulose I or cellulose IVI (Isogai et al., 1989). In those carbon atoms of the transgenic plants, the higher ®eld resonance became less emphasized than that in the wild-type plant as shown in Figure 4. The higher ®eld resonances are thought to originate from more mobile chains than that in crystal-core, for instance surface chains that can be accessible to water (Larrson et al., 1997). Therefore, these results indicate that the overexpression of poplar cellulase resulted in the decrease in the mobile chains of 1,4-b-glucans.

Discussion Cell wall modification We have inferred that the transgenic expression of poplar cellulase decreases the disordered 1,4-b-glucan on cellulose micro®brils within the wall. The hypothesis of better order among the micro®brils in the transgenic plants is supported by the NMR spectra. Previously, Horii et al. (1983) proposed a linear relationship between the 13C NMR chemical shifts of C6 and the torsion angles about the 1,4-b-glucosidic linkages. Three preferred conformations, gauche-gauche, gauche-trans, and trans-gauche, were correlated to the chemical shifts from 61 to 62, 62.7 to 64.5 p.p.m., and to about 66 p.p.m., respectively. They considered the chemical shift at around 66 p.p.m. to re¯ect a crystalline contribution with C6 in the trans-gauche conformation and the shift at 63 p.p.m. to re¯ect a surface or paracrystalline contribution with C6 in the gauche-trans conformation. According to their interpretation, the NMR spectra from the cell wall preparations can be explained by the transgenic plants having an increased proportion of trans-gauche conformation at the C6 carbon of 1,4-b-glucan (Figure 4), and hence a greater proportion of crystalline cellulose. We consider that overexpressing the poplar cellulase modi®ed cell walls by trimming off disordered glucose chains from the micro®brils. Bulk degradation of cellulose is unlikely because the amount of cellulose is nearly the same in the transgenic and wild-type plants. The amount of released cello-oligosaccahrides is a thousandth of the amount of cellulose but may exert a proportionate effect on cell wall properties through interactions with the cell wall matrix. It is possible that the increase in cello-oligosaccharides in the transgenic plants is due to an increase in sitosterol cello-oligosaccharides as a result of the activation of cellulose biosynthesis (Peng et al., 2002), but we believe

1103

that this is unlikely. The trimming of micro®brils by the enzyme is expected to solubilize some xyloglucan that was intercalated within disorderd paracrystalline domains of the micro®brils. This prediction is borne out by chemical analysis (Table 3), which showed that the transgenic plants contained less xyloglucan bound to cellulose micro®brils and more soluble xyloglucan. Enhancement of growth Transgenic A. thaliana overexpressing poplar cellulase (PaPopCel1) increased the size of all organs examined, including petiole and blade of rosette leaves and hypocotyls of etiolated seedlings. Similarly, transgenic Populus tremula overexpressing Arabidopsis cellulase (cel1) had longer internodes and longer ®ber cells (Shani et al., 1999). The cell enlargement by plant cellulases is probably related to a decrease in wall pressure and is unrelated to the osmolarity of cell solutes (Table 2). Based on the relative load±extension curves, a cross-linking component appears to be reduced in the walls of the transgenic plants compared to the wild-type plant. It is likely that the decreased cross-linking component and the increased wall plasticity are attributable to a decreased amount of tethering xyloglucan, which in turn accelerates growth in the transgenic plants (Figure 1 and Table 1). This agrees with a previous ®nding that the integration of xyloglucan oligosaccharides into pea stem segments solubilizes endogenous xyloglucan in the wall, weakens the cell wall, and accelerates elongation (Takeda et al., 2002). Poplar cellulase is expressed differently in A. thaliana than it is in the growing poplar leaf. In the poplar leaf, the expression of two cellulases corresponds to cellulose synthesis during growth (Ohmiya et al., 2002). Poplar cellulases may act in the expanding tissues, where cellulose synthesis is required. In the transgenic A. thaliana, the gene product appears to act in both expanding and mature tissues. Nevertheless, the transgenic poplar line (S7) overexpressing poplar cellulase produced about a 1.28-fold larger leaf than the wild-type poplar (Ohmiya et al., 2002). This phenomenon was attributed to about a twofold increase in the speci®c activity of cellulase, and a relationship between increased cellulase activity and tissue enlargement that is relatively similar to transgenic Arabidopsis. Therefore, we argue that poplar cellulases promote the expansion growth in transgenic poplar by trimming disordered 1,4-b-glucans. The trimming action may be related to the action of expansin, which disentangles the micro®brils glued by hydrogen-bonding interactions between cellulose and xyloglucan. It is also likely that overexpressed expansin activates endogenous cellulases by the increased activity of expansin (Cho and Cosgrove, 2000) because expansin enhances cellulase action (Cosgrove et al., 1998).


1104 Yong Woo Park et al. Enhancement of cellulose deposition Overexpression of poplar cellulase in A. thaliana caused the acceleration of cell enlargement and resulted in an increase in total cellulose content in the leaf. The content of total cellulose per total plant leaves was increased by the overexpression by a factor of 2±3, although the amount of cellulose per dry weight was constant (Table 3). If the disordered chains of 1,4-b-glucans cause the restriction of cell wall extension, the relaxation by trimming such disordered chains could result in tissue enlargement. If cellulose formation is restricted by the entanglement of xyloglucan and cellulose, the relaxation by trimming disordered chains may accelerate cellulose biosynthesis during the assembly of micro®brils. We propose that the above two relations are probably involved in the enhancement of cell growth and cellulose deposition in the transgenic Arabidopsis and poplar. Linkages between xyloglucan and cellulose The binding of xyloglucan to cellulose can be expressed by Langmuir adsorption isotherms, in which the polysaccharide binds as a monolayer to the surface of micro®brils (Hayashi et al., 1994). Analysis of the binding capacity for cellulose micro®brils of different surface area showed that the capacity was dependent on the surface area of the micro®brils. However, the native xyloglucan±cellulose complex (4% KOH-insoluble fraction) obtained here (Table 3) contains potentially much higher levels of xyloglucan than those in the potentially reconstructive complex; i.e. the maximal adsorption of xyloglucan to cellulose is 167 mg mg 1 cellulose composed of 36 chains (Hayashi et al., 1994). Xyloglucans are tightly bound to cellulose by hydrogen bonding in the growing walls of Arabidopsis leaf, where the polysaccharides are intercalated into micro®brils to cause disordered 1,4-b-glucan chains (Hayashi, 1989; Pauly et al., 1999). Even some xyloglucan might be woven deeply into the center of micro®brils because all the xyloglucan molecules were not solubilized from the cellulose micro®brils by the overexpression of poplar cellulase. We con®rmed that there is a xyloglucan tether, part of which is intercalated into cellulose micro®brils. This xyloglucan tether contributes to make the wall rigid, and the dissolution of the tether contributes to the loosening of the cell wall. Experimental procedures Plant materials Arabidopsis thaliana (ecotype Columbia) plants were grown in soil at 228C under 16 h : 8 h light:dark conditions. For selection of transgenic plants, seeds were sterilized in ethanol and bleach, rinsed with water, and germinated on agar medium containing 30 mg ml 1 kanamycin and the kanamycin-resistant seedlings

were planted in soil. Seventeen independent homozygote transgenic lines with PaPopCel1 cDNA were divided into kanamycin-resistant and -susceptible lines at a 3 : 1 ratio, which indicate T-DNA insertion at a single locus in the genome. From these lines, we selected four T3 lines for further investigation and used T2 progenies for histogram analysis of rosette. For determining the length of hypocotyls, seeds were germinated on nutrient medium as described by Estelle and Somerville (1987) with 1% sucrose in a growth chamber. Plates were wrapped in two layers of aluminum foil, and days after transfer of the plates to the growth chamber were counted.

Transgene constructs PopCel1 cDNA fragment was excised from pBluescript SK by digestion with BamHI and KpnI (Nakamura et al., 1995). The GUS-coding sequence of pBE2113 was removed from the fragment by digestion with BamHI and SacI, and the cDNA fragment was inserted in the pBE vector between the cauli¯ower mosaic virus 35S promoter and the Agrobacterium tumefaciens nos transcription terminator. The chimeric construct was introduced into disarmed A. tumefaciens strain LBA4404, which was used to transform A. thaliana by vacuum in®ltration.

Fractionation of apoplastic, symplastic, and wall-bound fractions After 30 days in the chamber, leaves were harvested and vacuumin®ltrated with de-mineralized water, blotted dry and weighed (Rohringer et al., 1983). The in®ltrated leaves were wrapped in aluminum foil, which was placed in a 15-ml tube, and centrifuged for 15 min at 440 g to yield the apoplastic solution. Cytoplasmic contamination as determined from the level of malate dehydrogenase (MDH, EC 1.1.1.37) activity (Bergmeyer and Bernt, 1974) was below the range of 0.2±0.5% in the solution compared with the total activity in the leaf homogenate. After extraction of apoplastic solution, leaves were homogenized in 20 mM sodium phosphate buffer (pH 6.2) in a mortar and the wall residue was washed thrice. The extract obtained was designated as the symplastic fraction. The wall-bound fraction was extracted from the wall residue with sample buffer (2% SDS, 10% glycerol, 100 mM DTT, 60 mM Tris±HCl, and 0.001% bromophenol blue, pH 6.8) at 808C for 10 min.

Determination of cello-oligosaccharides About 0.2 ml of apoplastic solution was boiled for 5 min and left at room temperature for 24 h to equilibrate the anomer con®guration between a- and b-types. The amount of cello-oligosaccharides was determined by using cellobiose dehydrogenase puri®ed from conidia spores of Phanerochaete chrysosporium (Samejima and Eriksson, 1992), according to the method previously reported (Tominaga et al., 1999). The reaction mixture contained 90 mU (10 ml) of cellobiose dehydroganase, 50 mM Cyt c (10 ml), and sample solution (70 ml) in 100 mM sodium acetate buffer, pH 4.2. After incubation for 5 min at room temperature, the absorbance at 550 nm was determined. A linear standard curve was obtained with a standard cellobiose solution and an absorbance of 0.5% corresponded to approximately 270 ng/100 ml of reaction mixture for cellobiose.

Histology For paradermal observations, leaves were ®xed in formalin, acetic acid, 70% ethyl alcohol (90 : 5 : 5, v/v) (FAA) solution and rendered transparent by incubation for several hours in a chloral hydrate


Overexpression of poplar cellulase solution composed of 200 g chloral hydrate, 20 g glycerol, and 50 ml water (Tsuga et al., 1996). Samples were observed under a microscope with Nomarski interference optics (Axioskop, Zeiss, Germany).

Determination of mechanical properties of leaf blade strip For a creep test, the leaf blade strip was immediately immersed in methanol at 658C for 10 min and transferred to fresh methanol at 48C. After re-hydration with 10 mM MES±NaOH buffer (pH 6.0) for 15 min at 08C, the samples were subjected to the creep-extension analysis using a Rheoner RE-33005 creep tester (Yamaden Co., Tokyo, Japan). The leaf strip (2 mm span) was subjected to the creep test to give a constant load (5 g) for 5 min, the load was immediately reduced to 0 g, and the shrinkage of the leaf strip was recorded as the ®nal length for 3 min. The residual extension after the shrinkage was de®ned here as plastic extension (Tanimoto, 1994; Tanimoto et al., 2000). As the creep extension during initial 5 min corresponded to 90% of extension after 60 min, we analyzed extension curve for 5 min. The load of 5 g was chosen as 65% of average breaking load for the leaf strips. The extension and breakage of a leaf blade strip (2 mm 10 mm) parallel to the midrib were examined by the tensile method (Ferry, 1970) using an automatic dynamic viscoelastometer TMA/SS6100 (Seiko Instruments, Tokyo, Japan). The native strip was clamped in 10 mM MES±KOH buffer (pH 6.2) at 238C. The experimental conditions were 0.5 g min 1 for load at 5 mm span. The tensile force was loaded in the longitudinal direction of leaf blade strips.

Measurement of osmotic concentration Osmotic concentrations of leaves were measured by the vapor pressure method (Miyamoto and Kamisaka, 1998). A leaf blade was excised from the rosettes and frozen at 208C. Frozen leaf was thawed at room temperature and centrifuged at 48C for 10 min at 1000 g. Aliquots (10 ml) of the supernatant were subjected to analysis in a vapor pressure osmometer (model 5520; Wescor, Logen, UT, USA). The measurement was repeated thrice and means were calculated.

Isolation of 4% KOH-insoluble fraction and carbohydrate contents Fresh leaves were freeze-dried to determine their dry weight. Then, the leaves were washed thrice with acetone and extracted thrice with 0.1 M sodium phosphate buffer (pH 6.2) to obtain cell wall preparations. The preparations were then extracted six times with 4% KOH/0.1% NaBH4 in an ultrasonic bath below 308C for 3 h to remove much polymeric material but retain most of xyloglucan and cellulose (Hayashi and Maclachlan, 1984). This wall residue was neutralized with 2 M acetic acid, washed with water and referred to as the `4% KOH-insoluble fraction'. The 4% KOH-soluble fraction was neutralized with 2 M acetic acid, dialyzed against water, and freeze-dried. The amounts of xyloglucan and cellulose were determined by methylation analysis because xyloglucan extracted from Arabidopsis leaf is precipitated during neutralization and dialysis (Zablackis et al., 1995). The amounts of terminal and 2-linked xyloses, terminal fucose, and 4,6-linked glucose are attributed to xyloglucan and that of 4-linked glucose to cellulose.

CP/MAS

13

C NMR

Cross-polarization magic angle spinning 13C NMR spectra were acquired on a JEOL CMX instrument operating at 74.66 MHz for

1105

13

C at an ambient temperature. Spin-locking ®elds for CP/MAS experiments were approximately 40 kHz with a contact time of 1 msec. Magic angle spinning speed was 5 kHz with an experimental re-cycle time of 5 sec. Typical re-cycling was done 10 000 times to obtain each spectrum.

Acknowledgements Dr T.I. Baskin is acknowledged for critically reading this manuscript. This work was supported by Research Grant BOP-03-II-1-1 from the Ministry of Agriculture, Forestry and Fisheries.

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Accession number: PopCel1, D32166.
