Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress

ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 39—47

www.elsevier.de/jplph

Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress A.K.M. Zakir Hossain, Hiroyuki Koyama, Tetsuo Hara Laboratory of Plant Cell Technology, Department of Biotechnology, Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu-shi, Gifu 501-1193, Japan Received 24 December 2004; accepted 16 February 2005

KEYWORDS Aluminum; Cell wall polysaccharides; Ferulic acid; p-coumaric acid; Triticum aestivum; Wheat

Summary The present study was conducted to investigate the cell wall properties in two wheat (Triticum aestivum L.) cultivars differing in their sensitivity to Al stress. Seedlings of Al-resistant, Inia66 and Al-sensitive, Kalyansona cultivars were grown in complete nutrient solutions for 4 days and then subjected to treatment solutions containing Al (0, 50 mM) in a 0.5 mM CaCl2 solution at pH 4.5 for 24 h. Root elongation was inhibited greatly by the Al treatment in the Al-sensitive cultivar compared to the Al-resistant cultivar. The Al-resistant cultivar accumulated less amount of Al in the root apex than in the Al-sensitive cultivar. The contents of pectin and hemicellulose in roots were increased with Al stress, and this increase was more conspicuous in the Al-sensitive cultivar. The molecular mass of hemicellulosic polysaccharides was increased by the Al treatment in the Al-sensitive cultivar. The increase in the content of hemicellulose was attributed to increase in the contents of glucose, arabinose and xylose in neutral sugars. Aluminum treatment increased the contents of ferulic acid and p-coumaric acid especially in the Al-sensitive cultivar by increasing the activity of phenylalanine ammonia lyase (PAL, EC 4.3.1.5). Aluminum treatment markedly decreased the b-glucanase activity in the Al-sensitive cultivar, but did not exert any effect in the Al-resistant cultivar. These results suggest that the modulation of the activity of b-glucanase with Al stress may be involved in part in the alteration of the molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar. The increase in the molecular mass of hemicellulosic polysaccharides and ferulic acid synthesis in the Al-sensitive cultivar with Al stress

Abbreviations: CWM, cell wall material; GLC, gas–liquid chromatography; PAL, phenylalanine ammonia lyase Corresponding author. Tel./fax: +81 58 293 2908. E-mail address: [email protected] (T. Hara). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.02.008

ARTICLE IN PRESS 40

A.K.M. Zakir Hossain et al. may induce the mechanical rigidity of the cell wall and inhibit the elongation of wheat roots. & 2005 Elsevier GmbH. All rights reserved.

Introduction The initial symptom of Al toxicity in plants is the inhibition of root elongation, which can be observed within a short time of exposure to Al (Ma et al., 2004). Despite decades of extensive efforts to elucidate the mechanism(s) of Al phytotoxicity, the primary cause of Al toxicity remains largely speculative and controversial (Delhaize and Ryan, 1995; Horst, 1995; Kochian, 1995; Matsumoto, 2000). When a plant grows under Al toxic conditions, the apoplast or cell wall of its roots is the first part to come in contact with Al. Aluminum strongly binds to the cell wall of root epidermal and cortical cells (Delhaize et al., 1993).The Al bound to the cell wall plays an important role in the inhibition of cell elongation (Zheng et al., 2003). Biochemical modifications of the cell wall, such as changes in the molecular size and quantities of cell wall polysaccharides, have been considered to be possibly involved in the regulation of cell wall extensibility (Kaku et al., 2002; Sakurai, 1991). Alteration of the molecular mass of the cell wall polysaccharides and mechanical extensibility of cell wall in response to various environmental stresses has been reported in various plant materials (Hoson, 1998). However, changes in the mechanical properties of the cell wall are still poorly characterized in wheat roots differing in their sensitivity to Al stress. Phenolic compounds are involved in many interactions of plants with their biotic and abiotic environments. These substances accumulate in different plant tissues and cells under the influence of various environmental stimuli. The major polysaccharidebound phenols in graminaceous are ferulate and p-coumarate, which are ester linked via their –COOH groups to specific –OH groups of wall matrix polysaccharides such as arabinoxylan (Nishitani and Nevins, 1988). Arabinoxylan chains esterified with ferulic acid can cross-link each other through diferulic acid bridges which are formed by peroxidase. These bridges lead to depression of cell elongation (Fry, 1979) and decrease the cell wall extensibility (Fry, 1986). It has been reported that Al increases the amount of wall-bound ferulic acid in wheat roots (Tabuchi and Matsumoto, 2001). But how Al affects the synthesis of ferulic acid is still not clear. The objective of this work was to investigate growth and modification of cell wall properties in

two wheat cultivars differing in their sensitivity to Al stress.

Materials and methods Root growth Seeds of Inia66 and Kalyansona wheat (Triticum aestivum L.) cultivars, which had been shown to be Al-resistant and Al-sensitive cultivars (Hossain et al., 2004), were surface sterilized with 5% sodium hypochlorite and stirred for 10 min to eliminate pest contamination. These seeds were washed and soaked with distilled water and kept in refrigerator for 24 h. The imbibed seeds were germinated on filter papers (Adventec Toyo, Japan) in Petri dishes containing 100 mM CaCl2 solutions. At 2 days after, the germinated seeds with a uniform root length of approximately 1–2 cm were transferred on slide mounts with floating nets, 15 plants/mount, and pre-cultured for 4 days in full nutrient solutions at pH 5.0.The solution was renewed every day with fresh nutrient solutions. All the experiments were conducted in a growth room at 25 1C under a 12 h light and 12 h dark regime, 70% relative humidity, 150 mmol/m2/s photon flux density during the 12 h day. The pH of the culture solution was closely monitored and adjusted in all the experiments during the treatment period. The full nutrient solution was composed of 500 mM NH4NO3; 500 mM Ca(NO3)2; 250 mM NH4NO3; 125 mM MgSO4; 2 mM KH2PO4; 2 mM FeCl3; 11 mM H3BO3; 2 mM MnCl2; 0.35 ZnCl2; 0.2 mM CuCl2 and 0.1 mM (NH4)6Mo7O4. Pre-culture solutions were replaced by treatment solutions containing Al (0, 50 mM) in a 500 mM CaCl2 solution at pH 4.5 with three replications. Treatment solutions were renewed every 12 h with fresh solutions. At 24 h after treatment the plants were harvested and the root length and fresh weight were measured. A portion of root tip (0–1 cm) was dried at 70 1C for 48 h for measuring Al contents of roots. Aluminum content of roots was determined by colorimetric method using pyrocatechol violet and hexamethylene tetramine buffer solution (Doughan and Wilson, 1974) after digesting plant samples with concentrated H2SO4 and H2O2. The results were expressed as mean values of measurements with replications7standard deviation (SD).

ARTICLE IN PRESS Modification of cell wall properties by Al-stress For collecting cell wall material (CWM), fresh root tip (0–1 cm) samples were instantly frozen in liquid nitrogen and stored at 80 1C in deep freezer.

Collection of Cell Wall Material Cell wall fractions were prepared by the method of Sakurai et al. (1987). A group of 90 root tips was fixed in boiling methanol for 10 min. The methanol extract was designated as a methanol-soluble fraction. Rehydrated root segments were homogenized in deionized water with a polytron homogenizer (PT 3000, Kinematica, Tokyo, Japan). The homogenate was centrifuged for 10 min at 1000g and then the residue was washed with deionized water, acetone and a methanol:chloroform mixture (1:1 v/v) and dried. The dried CWM was treated for 2 h with 2 units/mL porcine pancreatic a-amylase (type 1-A sigma) in Na-acetate buffer (pH 6.5, 50 mM) to remove starch and then for 18 h with 200 mg/mL pronase (Actinase; Kaken Kagaku Co., Tokyo, Japan) in Na-phosphate buffer (pH 6.5, 50 mM) containing 5% ethanol to remove proteins. The pectic substance was extracted from CWM by three treatments with 50 mM ethylenediaminetetra acetic acid at 95 1C for 15 min. The hemicellulosic polysaccharides were extracted for 18 h with 17.5% NaOH containing 0.02% NaBH4. The hemicellulosic fraction was neutralized with glacial acetic acid. The pectic and neutralized hemicellulosic fraction were dialyzed against deionized water for 36 h and lyophilized with freeze drier. The alkali-insoluble residue was first hydrolyzed with trifluoracetic acid (2 M, 121 1C, 1 h) to release remaining part of hemicellulosic substances (Yeo et al., 1995), and then washed with ethanol, ethyl acetate (1:1, v/v), dried in air under a fume hood and designated as cellulose.

Total sugar and uronic acid analysis Total sugar content of pectic and hemicellulosic fraction was quantified by the phenol–sulfuric acid method (Dubois et al., 1956) using glucose as standard. Uronic acids were measured by modified carbazole–sulfuric acid method (Dische, 1962) using galacturonic acid as standard.

Determination of the molecular mass of hemicellulosic polysaccharides Gel chromatography of the lyophilized hemicellulosic fractions in each treatment was carried out on a Sepharose CL-6B column (90 cm  2.5 cm) which had been equilibrated with 0.05 M phosphate

41 buffer (pH 7.8). The column had been calibrated with authentic dextrans (10, 40, 70, 120 and 510 kDa) purchased from Pharmacia and Sigma Chem. Co. The samples (ca. 5 mg) were dissolved in 5 mL of the 0.05 M phosphate buffer (pH 7.8).The insoluble impurity in the solution was removed by centrifugation at 1000g for 30 min. The supernatant was applied on a column and eluted with the same buffer at a flow rate of 20 mL/h. The elute was collected in 5 mL fractions and subjected to determination of total sugar contents as described above.

Gas–liquid chromatography The neutral sugars of pectin and hemicellulose were analyzed by gas–liquid chromatography (GLC). Acetylating of the sugars after conversion to alditol acetate was performed according to Blakeney et al. (1983). GLC was carried out on a Shimadzu GC-18A apparatus equipped with a flame ionization detector. A capillary column (25 m, 0.22 mm i.d., 0.25 mm Hicap CPB10) was used and operated at 220 1C with gas flow rate of 60 mL/min of nitrogen. Peak areas were measured with a Shimadzu Chromatocorder-21.

Analysis of phenolic compounds The phenolic compounds in each treatment were analyzed by alkaline hydrolysis and subsequent analysis by GLC. The root tips (0–1 cm) tissues were extracted with 80% ethanol for 3 h. After air drying, extracted tissues were macerated and ground in a mortar and pestle with liquid nitrogen. The sample (20 mg) was treated with 5 mL of 0.5 M NaOH at room temperature for 24 h under argon. A solution of vanillic acid in ethanol (4 mg/mL; 200 mL) was added as an internal standard and the mixture was acidified with 6 M HCl to pH 1.0. The solution was then extracted three times with ethyl acetate and the ethyl acetate soluble fraction was recovered. This fraction was dehydrated overnight with anhydrous sodium sulfate. The solution was then evaporated to dryness and the residue was dissolved in 200 mL of pyridine. A 20 mL aliquot of the pyridine solution was mixed with the same volume of N,O-bis (trimethylsilyl)-tri-fluoroacetamide and, after incubation for 30 min at 60 1C, the silylated sample was analyzed by GLC on a capillary column (model CBP-1, Shimadzu GC-18A). The column temperature was 100 1C initially and after 5 min, it was increased to a final temperature of 280 1C at a rate of 5 1C/min. The amount of ferulic acid and

ARTICLE IN PRESS 42

b-glucanase activity The apical (0–1 cm) root tips were excised and were immediately frozen with liquid nitrogen and kept at 80 1C until use. Extraction and assay of b-glucanase activity were carried out essentially by the methods of Chen et al. (1999). The frozen root samples (ca. 400 mg in fresh weight) were homogenized with ice cold 10 mM sodium phosphate buffer (pH 7.0). The homogenate was filtered through propylene mesh (32 mm). The fraction was washed with the same buffer and then suspended in 10 mM sodium phosphate buffer (pH 6.0) containing 1 M NaCl. The suspension was kept for 24 h at 4 1C and filtered through propylene mesh. The filtrate was used as enzyme extract for the measurement of b-glucanase activity. The reaction mixture (total 100 mL) contained 50 mg of b-glucans (b-1,3;1,4-Dglucans) and 5 mg of cell wall protein in 10 mM sodium phosphate buffer (pH 6.0). The mixture of solution was incubated for 6 h at 37 1C. After the incubation the reaction was terminated by boiling. Enzyme activity was determined by monitoring the increase in reducing sugars liberated from bglucans by the Somogyi–Nelson method (Somogyi, 1952) and expressed as glucose equivalent. Protein content was determined with a protein Assay Kit (Bio-Rad, Hercules, CA, USA).

PAL activity The crude enzyme preparation for phenylalanine ammonia lyase (PAL) was obtained by homogenizing 0.15 g of fresh tissue in 15 mL of an extraction medium containing 20 mM b-mercaptoethanol, 0.1 M sodium borate buffer, pH 8.8, and 5% (w/v) insoluble polyvinylpyrrolidone. After filtration with four layers of cheesecloth, the homogenate was centrifuged at 12000g for 20 min. The enzyme activity was determined by adding 1 mL of the crude enzyme extract to a reaction medium containing 1 mL of 0.2 M sodium borate buffer, pH 8.8 and 1 mL of 0.1 M L-phenylalanine. After incubation for 1 h at 30 1C, the reaction was stopped by adding 0.1 mL of 6 N HCl and the absorbance was determined at 290 nm (Cahill and McComb, 1992). PAL activity was calculated using the molar extinction coefficient of 104 mM/cm.

Root elongation [cm (24 h)-1]

Enzyme assays

1.8 1.6

b

b

b

1.4 1.2 1 0.8

a

0.6 0.4 0.2 0

Al content (mg kg-1 root DW)

p-coumaric acid was calculated as mg/mg root FW in all the treatments.

A.K.M. Zakir Hossain et al.

2500 c 2000 1500

b

1000 500 a 0

a Control

Al Treatment

Figure 1. Effects of Al treatment on root elongation and Al content of root tips of two wheat cultivars, Altolerant, & Inia66; Al-sensitive, ’ Kalyansona. Fourday-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test); vertical bars indicate the SD of means of three replications.

Results Root elongation Root elongation was inhibited by the Al treatment by about 11% for Inia66 and 54% for Kalyansona (Fig. 1). The Al content in root apices (0–1 cm) was higher in Kalyansona than in Inia66 (Fig. 1).

Cell wall polysaccharides Aluminum treatment significantly increased the contents of pectin and hemicellulose in the cell wall of roots in Kalyansona while having little effect in Inia66 (Table 1). Aluminum treatment had no effect on the content of cellulose in both the cultivars.

Molecular mass of hemicellulosic polysaccharides The molecular mass distribution of hemicellulosic polysaccharides in the cell wall of Inia66 and Kalyansona was determined by phenol–sulfuric acid method on gel permeation chromatography

ARTICLE IN PRESS Modification of cell wall properties by Al-stress Table 1. cultivars

43

Effects of Al treatment on the pectin, hemicellulose and cellulose contents in root tips of two wheat

Treatment

Inia66 (mg/mg root FW)

Control Al

Kalyansona (mg/mg root FW)

Pectin

Hemicellulose

Cellulose

Pectin

Hemicellulose

Cellulose

1.38a 1.50a

14.25a 15.50a

10.50a 9.50a

1.68a 2.05b

18.56a 23.50b

16.80a 15.25a

Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test).

Vo

0.5

510

Inia66

0.4

70

10

Control

Vt Al

0.3

the root growth of Kalyansona by changing the hemicellulose properties. We checked the statistical significance of average molecular mass of the data shown in Fig. 2. We found that the average molecular mass of Al-treated roots was significantly higher than that of control (data not shown).

Absorbance at 490 nm

0.2

Sugar compositions

0.1 0 Kalyansona 0.5 0.4 0.3 0.2 0.1 0

20

30

40

50 60 Fraction No.

70

80

Figure 2. Elution profiles of hemicellulosic polysaccharides of root tips in Al-tolerant, Inia66 and Al-sensitive, Kalyansona wheat cultivars. Gel chromatography of the lyophilized hemicellulosic fractions in each treatment was carried out on a Sepharose CL-6B column (2.5 cm  90 cm) which had been equilibrated with 0.05 M phosphate buffer (pH 7.8). The column had been calibrated with authentic dextrans (10, 40, 70, 120 and 510 kDa). Total sugar content in each fraction was determined by the phenol–sulfuric acid method. Vertical bars of X-axis denote the elution position of molecular mass standards (in kDa) and void volume (Vo). Each elution profile is means of three replications without SD.

(Fig. 2). Hemicellulosic polysaccharides of Inia66 with and without Al stress were eluted in the similar molecular mass regions, while those of Kalyansona with Al stress were characterized by higher molecular masses compared to those without Al stress. In fact, the elution profile peak with control treatment was fraction 52, but this shifted to 45 with Al treatment in the Al-sensitive cultivar (Fig. 2). It is inferred that Al treatment affected

The results of neutral sugars of pectin and hemicellulose for the Al-resistant cultivar, Inia66 did not differ between Al treatment and control and therefore, the data are not shown. The neutral sugar part of pectin fraction was composed of xylose, glucose, arabinose, galactose, and fucose (Table 2). Aluminum treatment did not exert any effect on the contents of neutral sugars in the Kalyansona except a modest increase in the content of glucose. Aluminum treatment increased the uronic acid content in pectin compared to the control. The neutral sugar part of hemicellulosic fraction was composed of mainly xylose, arabinose, glucose and galactose (Table 3). Aluminum treatment remarkably increased the contents of glucose, xylose and arabinose in comparison with those without Al stress in the Kalyansona and the glucose which may be present as b-glucan level in hemicellulose appears to be considerably larger than xylose and arabinose. Fucose and galactose appear to be depressed slightly in the presence of Al. The arabinoxylan content was calculated as the combined amount of arabinose and xylose present in the cell wall. The arabinoxylan content was higher in the Al treatment than in the control. Aluminum treatment had no influence on the contents of uronic acid in hemicellulose (Table 3).

Phenolic compounds Aluminum treatment significantly increased the contents of ferulic acid and p-coumaric acid in Kalyansona compared to those without Al stress but had a smaller effect in Inia66 (Table 4). Ferulic acid

ARTICLE IN PRESS 44 Table 2. cultivar Treatment

Control Al

A.K.M. Zakir Hossain et al. Effects of Al treatment on sugar composition of pectin fraction in root tips of Al-sensitive, Kalyansona Neutral sugar composition (mol%)a

Uronic acid (%)b

53.4a 56.3b

Fuc

Ara

Xyl

Man

Gal

Glc

9.09a 8.16a

16.45a 16.36a

30.47a 30.66a

1.15 ND

14.22a 14.07a

27.25a 32.95b

Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Pectin fraction was hydrolyzed with TFA and subjected to the determination of neutral sugar composition by GLC. Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose. Uronic acids were determined by Carbazole–sulfuric acid method. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test). ND—not detected. a Each value is the mean of three replications and for any value the error was less than 5%. b Estimated in terms of galacturonic acid.

Table 3. Treatment

Control Al

Effects of Al treatment on sugar composition of hemicellulosic fraction in root tips of Kalyansona cultivar Neutral sugar composition (mol%)b

Uronic acid (%)a

12.3a 12.0a

Rha

Fuc

Ara

Xyl

Gal

Glc

Unknownc

Arabinoxyland

0.68 ND

3.30a 2.84a

19.95a 21.25b

54.6a 56.1b

7.52a 6.31a

10.71a 14.43b

3.19 —

74.57a 77.38b

Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Pectin fraction was hydrolyzed with TFA and subjected to the determination of neutral sugar composition by GLC. Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose. Uronic acids were determined by carbazole–sulfuric acid method. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test). ND—not detected. a Estimated in terms of galacturonic acid. b Each value is the mean of three replications and for any value the error was less than 5%. c In each treatment, the identities of some unknown components were not confirmed. d Arabinoxylan was calculated as the combined amount of arabinose and xylose.

Table 4. Effects of Al treatment on the contents of ferulic and p-coumaric acid in the cell wall of root tips in two wheat cultivars Treatment

Inia66 (mg/mg root FW)

Kalayansona (mg/mg root FW)

p-coumaric acid

Ferulic acid

p-coumaric acid

Ferulic acid

1.09a 1.13a

4.30a 5.65b

1.20a 1.75b

5.25a 8.45b

Control Al

Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM ) AlCl3 for 24 h at pH 4.5. Phenolic compounds were released from cell wall by alkaline hydrolysis and were extracted with ethyl acetate after acidifying to pH 1.0 with 6N HCl. Ferulic acid and p-coumaric acid were determined using GC system with CBP-1 column. Means with different letters are significantly different (P40.05, Duncan new Multiple Range Test).

was the predominant phenolic compound in the cell walls of wheat roots.

without Al stress (Fig. 4). Aluminum treatment increased PAL activity in the roots of Inia66 and Kalyansona by about 12% and 43%, respectively.

Activity of enzymes The activity of b-glucanase was decreased significantly with Al stress in Kalyansona and the activity was not affected significantly in Inia66 compared to those without Al stress (Fig. 3). Inia66 showed higher PAL activity than in Kalyansona

Discussion In the present study, we found that the Inia66 cultivar showed higher root elongation and accumulated less Al with Al stress in the roots compared

ARTICLE IN PRESS Modification of cell wall properties by Al-stress 4.5 µg Glc mg-1 root FW

b

b

b

3.5 3

a

2.5 2 1.5 1 0.5 0 b

b

1.4 µg Glc µg-1 protein

β-glucanase activity

4

b

1.2

a

1 0.8 0.6 0.4 0.2 0

Control

Al Treatment

Figure 3. Effects of Al treatment on b-glucanase activity in root tips of two wheat cultivars, Al-tolerant, & Inia66; Al-sensitive, ’ Kalyansona. Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Enzyme activity was determined by monitoring the increase in reducing sugars liberated from b-glucans by the Somogyi–Nelson method (Somogyi, 1952) and expressed as glucose equivalent. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test); vertical bars represent the SD of means of three replications.

PAL activity (µmol min-1 g -1 FW )

8 b

7 6 5

ab ab a

4 3 2 1 0

Control

Al Treatment

Figure 4. Effects of Al treatment on PAL activity in root tips of two wheat cultivars, Al-tolerant, & Inia66; Alsensitive, ’ Kalyansona. Four-day-old seedlings were exposed to 0.5 mM CaCl2 solution containing (0, 50 mM) AlCl3 for 24 h at pH 4.5. Means with different letters are significantly different (P40.05, Duncan New Multiple Range Test); vertical bars represent the SD of means of three replications.

45 to the Kalyansona cultivar (Fig. 1). Several mechanisms for Al exclusion have been proposed, including exudation of chelate ligands, formation of a plant-induced pH barrier in the rhizosphere or root apoplasm, immobilization of Al at the cell wall, selective permeability of the plasma membrane and Al efflux (Taylor, 1991; Kochian, 1995). In addition, it has been suggested that binding of Al to the apoplast (cell wall) may be involved in Al toxicity (Horst, 1995). The finding that pectin and hemicellulose contents in the cell wall of Al-sensitive cultivar increased with Al stress (Table 1) is consistent with reports on squash roots by Le Van et al. (1994) and wheat roots by Tabuchi and Matsumoto (2001). The increase in pectin and hemicellulose contents by the Al treatment might be the result of either the stimulation of synthesis or the inhibition of degradation of cell wall polysaccharides. Aluminum treatment had no influence on the cellulose content of root tips (0–1 cm) in both the cultivars. It indicates that Al is not affecting in the synthesis of cellulose primarily in the elongating regions. The amount of Al binding to pectin may affect physical properties of the cell wall and this amount depends on the pectin content present in the root tips. A higher content of pectin in the Al-sensitive cultivar (Table 1) may indicate the possibility of larger amount of Al binding. However, the influence of pectin on the rigidity of cell wall in wheat roots may be small because of very low content of pectin in the cell wall (Table 1). Hemicellulosic polysaccharides have been considered as candidates for determinants of the mechanical properties of the cell wall in graminaceous plants because they are the major constituents of the matrix fraction of the cell wall and form linkages within the cell wall architecture (Sakurai, 1991; Carpita and Gibeaut, 1993). In addition to the content of cell wall polysaccharides, their chemical and physical properties play an important role in the regulation of the mechanical properties of cell wall. The increase in glucose content in pectin (Table 2) in the Alsensitive cultivar with Al stress may be due to the increase in callose content as reported by Wissermeier et al. (1987). The molecular mass of hemicellulosic polysaccharides is related to the viscous state of cell wall and involved in the regulation of mechanical extensibility of cell walls in graminaceous plants (Masuda, 1990; Sakurai, 1991; Hoson, 1998). Aluminum treatment clearly increased molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar (Fig. 2). The neutral sugar composition of hemicellulose (Table 3) indicates that xylose, arabinose and

ARTICLE IN PRESS 46 glucose are the main sugars. These sugars are released from arabinoxylan and b-glucan which are the main hemicellulosic polysaccharides of cereals (Darvill et al., 1980; Shibuya, 1984). Some xylose is also originated from xyloglucan and some arabinose from glycoprotein, but xyloglucan and arabinogalactan (Glycoprotein) content is only minute in monocotyledonous species (Bacic et al., 1988; Obel et al., 2002). Therefore, the total amount of arabinose and xylose (Table 3) can be considered to be the content of arabinoxylan in the cell wall. These ideas suggest that the high molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar by the Al treatment (Fig. 2) may be related to the decreases in arabinoxylan and b-glucan degradation in the cell wall, and that the b-glucan may be eluted in high molecular mass regions on the gel permeation chromatography (Sakurai and Kuraishi, 1984). The results of b-glucanases activity clearly showed that Al treatment decreased the activity of this enzyme in the Al-sensitive cultivar (Fig. 3). The decrease in the activity may increase molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar (Fig. 2) by inhibiting metabolic turnover of the b-glucan. The cross-linking of phenolic compounds with arabinoxylan in the cell wall of cereals also provides mechanical strength and extensibility of the cell wall (Abdel-Aal et al., 2001). Ferulic acid esters act as starting material to produce lignin and cross-linking substances with hemicellulose during plant growth and development (Ralph et al., 1995). The increase in ferulic acid and p-coumaric acid contents in the cell wall (Table 4) suggests that the specific activity of enzymes in the phenylpropanoid pathway may be promoted by the Al treatment for the biosynthesis of ferulic acid, p-coumaric acid and also lignin. In fact, the activity of PAL was increased remarkably by the Al treatment, especially in the Al-sensitive cultivar (Fig. 4). Aluminum may affect on the activity of PAL which plays a key role in the biosynthesis of ferulic acid and pcoumaric acid in phenylpropanoid pathway. So, the greater increase in the activity of PAL with Al stress may accumulate more amounts of phenolic compounds and the activation of this enzyme may be related to the sensitivity of Al. It is concluded that the higher content of ferulic acid in the cell wall in the Al-sensitive cultivar with Al stress may form extensive cross-linking with arabinoxylan and inhibit root growth. Aluminum treatment may decrease the b-glucan degradation by decreasing the b-glucanase activity, causing an increase in the molecular mass of hemicellulosic polysaccharides, leading to increase the cell wall rigidity. Taken together b-glucan, arabinoxylan and ferulic acid play

A.K.M. Zakir Hossain et al. a central role in both growth regulation and responses to Al stress of wheat plants.

Acknowledgements We express our hearty thanks to Prof. Ryo Yamauchi for his invaluable suggestions to determine ferulic acid and p-coumaric acid. We would like to thank Dr. Onwona-Agyeman Siaw, Gifu University for his critical reading and English editing of this manuscript. The study was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

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