Phenylpropanoids as a Protectant of Aluminum Toxicity in Cultured ...

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and Fe(II), indicating that the phenylpropanoids acted as antioxidant molecules. ... the phenylpropanoids protect tobacco cells from cytotoxic lipid peroxidation ...
Plant Cell Physiol. 39(9): 950-957 (1998) JSPP © 1998

Phenylpropanoids as a Protectant of Aluminum Toxicity in Cultured Tobacco Cells Yoko Yamamoto 1 2

1>3

, Akiko Hachiya 1 , Hiroki H a m a d a 2 and Hideaki Matsumoto '-4

Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki, 710 Japan Department of Applied Science, Okayama University of Science, 1-1 Ridai-cho, Okayama, 700-005 Japan

Aluminum (Al) enhances ferrous ion [Fe(II)]-mediated peroxidation of lipids, which is lethal to normal tobacco cells, but not to phosphate (PJ-starved cells ( —P cells). We found that tobacco cells accumulated phenylpropanoid compounds including chlorogenic acid (CGA) and caffeic acid (CA) during P, starvation. The accumulation was inhibited by 2-aminoindan-2-phosphonic acid (AIP), a specific inhibitor of L-phenylalanine ammonia lyase (PAL). CGA, CA and also an extract containing the phenylpropanoid compounds from — P cells protected normal cells ( + P cells) efficiently from both lipid peroxidation and the loss of viability caused by the combined application of Al and Fe(II), indicating that the phenylpropanoids acted as antioxidant molecules. — P cells exhibited approximately 25-fold higher specific activity of PAL than + P cells. The content of the phenylpropanoids and the activity of PAL were not affected by the combined treatment with Al and Fe(II) in either + P cells or — P cells. These results suggest that an increase in PAL activity during P, starvation enhances the accumulation of phenylpropanoids, and that the phenylpropanoids protect tobacco cells from cytotoxic lipid peroxidation caused by the combination of Al and Fe(II). Key words: Aluminum — Lipid peroxidation — Nicotiana tabacum — Phenylpropanoid — Phenylalanine ammonia lyase — Phosphate starvation.

Al is considered to be a major factor causing the inhibition of plant root growth in acid soils. However, the primary lesion leading to root-growth inhibition has not yet been fully elucidated (for reviews, see Taylor 1988, Rengel 1992, 1996, Kochian 1995, Horst 1995). Al has the stimulatory effect on the Fe(II)-mediated peroxidation of lipids in phospholipid liposomes, which occurs nonenzymatically (Gutteridge et al. 1985, Oteiza 1994,

Verstraeten et al. 1997). Verstraeten et al. (1997) reported evidence suggesting that Al 3+ and Al3+-related cations (Sc 3+ , Ga 3+ , In 3+ , Y 3+ , La 3+ , Be2+) without redox capacity can stimulate the Fe(II)-initiated peroxidation of lipids by increasing lipid packing and by promoting the formation of rigid clusters. Both processes may bring phospholipid acyl chains closer together, thus favoring the propagation step of lipid peroxidation. In nutrient medium, Al alone is not apparently toxic to cultured tobacco cells, whereas the combined application of Al and Fe(II) causes cell death and the peroxidation of lipids (Ono et al. 1995). Since lipophilic antioxidants prevent both the peroxidation of lipids and the loss of viability caused by the presence of Al and Fe(II) together, the peroxidation of lipids seems to cause irreversible membrane damage and subsequent death in tobacco cells (Yamamoto et al. 1996, 1997). P r starved tobacco cells ( —P cells) tolerate the toxicity of the combination of Al and Fe(II), without increasing the peroxidation of lipids (Yamamoto et al. 1996). This suggests that — P cells accumulate antioxidants to protect the plasma membrane from the radical-chain reactions in the peroxidation of lipids. Because ^-carotene accumulates in tobacco cells during Pj starvation, /^-carotene is one of the candidates for a radical-trapping antioxidant in the plasma membrane in — P cells (Yamamoto et al. 1996). However, it is probable that other antioxidant molecules (e.g., vitamin E, glutathione, phenylpropanoids) (Halliwell and Gutteridge 1989) also participate in the prevention of lipid peroxidation in — P cells. In our preliminary experiments with tobacco cells, vitamin E (a-tocopherol) and glutathione existed at the same levels in + P cells and — P cells (6-d starvation). Therefore, in this study, we examined the accumulation of phenylpropanoid compounds during Pj starvation, and their role in protecting cells from the peroxidation of lipids caused by Al and Fe(II) together. Materials and Methods Tobacco cells, medium and culture conditions—The nonchlorophyllic tobacco cell line, SL, derived from Nicotiana tabacum L. cv. Samsun (Nakamura et al. 1988) was used in this study. Cells were grown in a modified version of Murashige-Skoog's (MS) medium (about pH 5.0 after autoclaving) on a rotary shaker operated at 100 rpm at 25°C in darkness (Yamamoto et al. 1994, 1996). For preparation of — P cells, cells at the logarithmic phase

Abbreviations: AIP, 2-aminoindan-2-phosphonic acid; CA, caffeic acid; CGA, chlorogenic acid; FAB, fast atom bombardment; Fe(II), ferrous ion; LC-MS, liquid chromatography-mass spectrometry; TBA, thiobarbituric acid; tR, retention time. 3 To whom correspondence should be addressed. Telephone and Fax: 086-434-1210, E-mail: [email protected] 4 CREST, Japan Science and Technology Corporation (JST). 950

Antioxidant effect of phenylpropanoids of growth ( + P cells) were transferred into Pi-free, modified MS medium, and cultured for various numbers of day as described previously (Yamamoto et al. 1996). AIP was kindly provided by Dr. N. Amrhein (Institute of Plant Sciences, Swiss Federal Institute of Technology, Switzerland). To inhibit the accumulation of phenylpropanoids during P-, starvation, cells were cultured in Pj-deprived medium in the presence of AIP (f.c. 100 |/M). Treatment with Al and determination of cell viability—Cells were treated with A1C13 or without A1C13 (control) in the presence of 100 ^iM FeSO4 in medium A [modified MS medium prepared without Pi and Fe(II)-EDTA], at pH 4.0, at a cell density of 100 mg fresh weight per 10 ml for 18 h on a rotary shaker operated at 100 rpm at 25°C in darkness (Yamamoto et al. 1994). Pj and EDTA were omitted from the treatment medium, because they have high affinity for Al and block the interaction of Al with cells (Yamamoto et al. 1995). After treatment, cells (from 10-ml aliquots of culture, corresponding to 100 mg fresh weight at the start of treatment) were harvested, washed and resuspended with Alfree medium (a modified MS medium) for post-treatment culture. Viability of Al-treated cells was estimated from the relative growth (the fresh weight of Al-treated cells relative to that of untreated control cells) after the post-treatment culture for 7 d. When cells were treated with Al in the presence of a phenylpropanoid compound (CGA, CA) or an extract containing phenylpropanoid compounds from — P cells, the compound (or the extract) was added to the suspension of cells at the start of the treatment, just prior to the addition of Al and Fe. CGA and CA were dissolved in DMSO, but the concentration of DMSO in the medium was 0.1% (v/v) or less. The phenylpropanoid compounds were extracted from — P cells with methanol containing \% HC1 as described below (see Extraction and analysis of phenylpropanoids). The extract was lyophilized, dissolved in DMSO, and then added to the treatment medium as described above. For the quantitation of total content of phenylpropanoids in the extract, the calibration coefficient of CA (OD32o 1 -0 is equivalent to ca. 56 /JM CA) was used. Quantitation of Al and Fe in cells—After treatment with or without Al, cells in 10-ml aliquots of culture were harvested, washed with medium A (pH 5.0) and then digested in a mixture of acids as described previously (Yamamoto et al. 1994). Al and Fe in digested samples were quantitated with a simultaneous multi-element atomic absorption spectrophotometer with a graphite furnace atomizer (model Z-9000; Hitachi, Tokyo, Japan). Assessment of the peroxidation of lipids—After treatment with or without Al, cells in 10-ml aliquots of culture were harvested, washed with sucrose-free medium A (pH 5.0), and then the peroxidation of lipids in cells was assessed by the TBA method, in which TBA-reactive substances was quantitated as malondialdehyde as an end product of lipid peroxidation (Ono et al. 1995). Extraction and analysis of phenylpropanoids—Cells (300 mg fr wt) were suspended in 1 ml of methanol containing 1% HC1. After one hour incubation at 25°C, the cell suspension was centrifuged for 10 min at 4,000 x g and the supernatant was filtered and used for analysis. The extract was fractionated by reverse-phase HPLC. The sample was loaded on a C ]8 column (Nakalai Cosmosil 5Ci8, 4.6x250mm, Nakalai Tesque, Inc., Kyoto, Japan) and the column was eluted at a flow rate of 0.6 ml min" 1 at 25CC. The mobile phase was _a mixture of ultra-pure water containing 0.5% of formic acid and 100% methanol (8 : 3, v/v). The eluate was monitored at 320 run. Each peak in the HPLC profile was identified and quantified by comparison of its retention time and its absorption spectrum, by reference to authentic compounds. The unidentified phenylpropanoid compounds (peaks 1,

951

2 and 3) eluted before peak 4 (CGA) (see Fig. 1), were quantified as CA, />-coumaric acid and ferulic acid, respectively, because of the similarity of their absorption spectra with the authentic chemicals (see Results and Discussion). For identification of peak 1, 2 and 3, LC-MS was obtained using a Fisons Instruments VG Analytical Auto-Spec-Q (MSI) equipped with FAB ionization (VG; London). LC conditions were as described above (see HPLC conditions). FAB-MS was carried out at an accelerating voltage of 8 kV and a resolution of 1 : 1,000. Glycerol cluster ions were used for calibration. The FAB gun was operated at 40 kV with cesium as the FAB gas. CGA, CA, /?-coumaric acid, ferulic acid were purchased from Nakalai Tesque Inc. (Kyoto, Japan). Assay of PAL—PAL activity was assayed by the spectrophotometric measurement of formation of cinnamic acid as described by Edwards and Kessmann (1992) with minor modifications. Cells (1 g fr wt) were disrupted in 2 ml of 50 mM Tris-HCl (pH 8.5) containing 14 mM 2-mercaptoethanol and 5% (W/V) polyvinylpyrrolidone, and centrifuged at 10,000 x g for 10 min at 4°C. Endogenous cinnamic acid in the cell extracts was removed twice with Dowex 1 as described. For assay of PAL activity, the cell extracts were incubated at 40°C with L-phenylalanine in 50 mM Tris-HCl (pH 8.5) with a parallel incubation of the sample with D-phenylalanine as control. The formation of cinnamic acid was monitored by taking absorbance readings at 290 nm at 30-min intervals for 2 h, and the molar extinction coefficient of cinnamic acid in 50 mM Tris-HCl (pH 8.5) was used for the quantification of cinnamic acid formed. Statistical analysis—Each experiment was repeated twice with similar results. All values are shown as the means±S.E. of triplicate results obtained in both experiments. Results and Discussion Accumulation of phenylpropanoid compounds during P, starvation in tobacco cells—Tobacco cells starved for Pj in Pj-free medium more than 4 d exhibit tolerance to the toxicity of Al and Fe given together as compared to the cells at the logarithmic phase of growth ( + P cells) (Yamamoto et al. 1996). In this experiment, lipophilic compounds were extracted with methanol containing \% HC1 from + P cells and — P cells that had been starved of Pj for 6 d. HPLC profiles of the extracts from + P cells revealed four main peaks (Fig. 1). — P cells exhibited increases in the amounts of these 4 peaks, and, in addition, a minor peak (peak 5). The peaks 4 and 5 were identified as CGA and CA, respectively, by both retention time and its absorption spectra in two different solvents (0.5% formic acid-methanol mixture, 8 : 3 , v/v; /z-butanol-methanol-acetic acidwater mixture, 1 : 5 : 2 : 92, v/v, Shinozaki et al. 1988). The major peaks (1, 2, 3) exhibited absorption spectra corresponding to CA, p-coumaric acid and ferulic acid, respectively (data not shown). LC-MS spectrometry of peak 1 ([M + H] + at m/z, 251; [ M - H ] - , 249), peak 2 ([M + H ] + , 235; [ M - H ] - , 233) and peak 3 ([M + H] + , 265; [ M - H p , 263) indicated that the molecular mass of each peak was 250 (peak 1), 234 (peak 2) and 264 (peak 3). The base peak at positive mass for each peak was 163 (peak 1), 147 (peak 2) and 177 (peak 3), suggesting an addition of the same resi-

952

Antioxidant effect of phenylpropanoids

111

+P cells

-p cells

£

3-

O)

U o

i 10

20

O

10

1-

20

Retention Time (min) Fig. 1 Profiles after HPLC of phenylpropanoid compounds from + P cells and — P cells. Phenylpropanoids were extracted from + P cells (A) and — P cells (6-d starvation) (B) and analyzed by reverse-phase HPLC as described in Materials and Methods. The column was eluted with a mixture of 0.5% formic acid and methanol ( 8 : 3 , v/v). The content of the numbered peaks were deduced as follows: 1, unidentified phenylpropanoid (a derivative of CA; t R , 6.4 min); 2, unidentified phenylpropanoid (a derivative of p-coumaric acid; tR, 7.9 min); 3, unidentified phenylpropanoid (a derivative of ferulic acid; tR, 9.0 min); 4, CGA (tR) 12.0 min); 5, CA(t R ) 15.9 min).

due of molecule mass of 87. Mizusaki et al. (1971) reported that tobacco cells growing in suspension culture predominantly accumulate caffeoyl-, p-coumaroyl- and feruloyl putrescine, whose molecular weights are the same as peak 1, 2 and 3, respectively. Furthermore, Knobloch and Berlin (1981) reported that the accumulation of cinnamoyl putrescines (caffeoyl putrescine, feruloyl putrescine) in Nicotiana tabacum cells in suspension culture is enhanced by Pi depletion of the culture medium. Thus, it is likely that the compounds at peaks 1, 2 and 3 may be the conjugated form with putrescine (1,4-butanediamine) of CA, pcoumaric acid and ferulic acid, respectively. Further analysis of the structures of these compounds has been performed in our laboratory. Figure 2 shows the amounts of peaks 1, 2, 3 and 4 (CGA) accumulated in — P cells (6-d starvation), which were calculated from molar extinction coefficients at 320 nm with references to CA (for peak 1), /?-coumaric acid (for peak 2) and ferulic acid (for peak 3), respectively. Peak 1 was a major peak accounting for 81% of the total compounds, whereas peak 2, 3 and 4 (CGA) were minor peaks accounting for 7, 10, and 2% of the total compounds, respectively. The accumulation of all these peak compounds was thoroughly inhibited when cells were cultured in Pj-deprived medium in the presence of AIP, a specific inhibitor of PAL (Zon and Amrhein 1992) (Fig. 2). Thus, all these compounds were considered to be phenylpropanoids synthesized by the action of PAL.

Fig. 2 The inhibitory effect of AIP on the accumulation of phenylpropanoid compounds in — P cells. + P cells were starved for P ; in the presence or absence of AIP in Pi-free modified MS medium for 6d. Phenylpropanoids were extracted from these cells and were analyzed by HPLC as shown in Figure 1. The amounts of the peaks were calculated from the molar extinction coefficient at 320 nm by references to CA (for peak 1), p-coumaric acid (for peak 2), ferulic acid (for peak 3) and CGA (for peak 4), respectively. Each peak value shows the mean±S.E. (n=3).

Figure 3 shows the accumulation kinetics of these compounds in tobacco cells during the Ps starvation for 9 d . Peaks 1,3, and 4 (CGA) showed a similar accumulation pattern; these peaks did not accumulate significantly until 3 d after start of Pj starvation, and then continuously increased until 9 d. On the other hand, the content of peak 2 increased linearly over the Pj starvation period. Phenylpropanoids protect cells from lipid peroxidation and from the loss of viability caused by the combination of Al and Fe(II)—Lipophilic antioxidants (N,N"diphenyl-p-phenylenediamine, butylated hydroxyanisol, propyl gallate) prevent efficiently not only the peroxidation of lipids but also the loss of viability and the accumulation of Al and Fe in tobacco cells treated with both Al and Fe(II) together in nutrient medium (Yamamoto et al. 1997). This suggests that most part of cell death and of the accumulation of Al and Fe are caused by the peroxidation of lipids. Therefore, firstly the protective effects of CGA (peak 4) on the peroxidation of lipids and the loss of viability in the + P cells treated with both Al and Fe(II) were examined (Fig. 4). CGA at 50 ^M completely prevented the peroxidation of lipids and also the loss of viability (Fig. 4). CGA is a CA conjugated with quinic acid, and CA also prevented both the peroxidation of lipids and the loss of the viability at the same dose range as CGA (data not shown). Thus, the protective effects of CGA seem to be attributed to the action of CA.

Antioxidant effect of phenylpropanoids

953

G 120 100 "

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ITS — O 58

The phenylpropanoid compounds extracted from — P cells (6-d starvation) also protected + P cells from the loss of viability caused by the combined application of Al and Fe(II), as efficiently as CGA. Figure 5 shows that the phenylpropanoid mixture at a concentration of OD320 1.0 [ca. 56 juM CA equivalent, see Materials and Methods] protected + P cells completely from death. The extract from the cells that had been starved for Pj for 6 d in the presence of AIP contained only a slight amount of phenylpropanoids (see Fig. 2), and did not protect + P cells from the loss of viability (data not shown). This suggests that the phenylpropanoids accumulated in — P cells actually protect + P cells from the loss of viability. The phenylpropanoid mixture extracted from — P cells also protected + P cells from both the peroxidation of lipids and the accumulation of Al and Fe due to the combined application of Al and Fe(II) (Table 1). In this table, + P

30

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50

T

(B)

^——-_

T

8060-

20

Fig. 3 The accumulation of phenylpropanoid compounds during P; starvation in cells. + P cells were starved for P( in P r free modified MS medium for up to 9 d. At indicated times, phenylpropanoids were extracted from the — P cells and were analyzed by HPLC as shown in Figure 1 and 2. Each peak value shows the mean±S.E. (n = 3).

20

a1

40-

0 3 6 9 Duration of Pi starvation (days)

10

J

00

T

y

/

/

—0

Al

—•—+AI

1

1

1

10 20 30 40 50 Cone, of CGA in medium (u.M)

Fig. 4 Inhibitory effects of CGA on the Al-enhanced peroxidation of lipids and on the Al-enhanced loss of viability in + P cells treated in nutrient medium that contained Fe(II). In the presence of various concentration of CGA, + P cells were treated with (•) or without (O) 150^M AlCl3 in medium A (pH 4.0) containing 100//M FeSO4 for 18 h at a cell density of 100 mg fr wt/lOml. Then the cells (from 10-ml aliquots) were collected, washed and the extent of lipid peroxidation (A) and viability (B) were determined as described in Materials and Methods. In (B), 100% viability corresponds to the post-treatment growth of control cells that had been treated without Al in the absence of CGA. All data show the means+S.E. (n=3).

cells of lOOmgfrwt were treated with Al in 10-ml treatment medium containing Fe(II) in the presence of the mixture of phenylpropanoids at OD320 1.0 (ca. 0.56//mol CA equivalent/10 ml). On the other hand, —P cells (6-day starvation) accumulated phenylpropanoid compounds at the concentration of 4.1//mol/300 mg fr wt (=1.3/miol/100 mg fr wt) (the total of peak 1,2,3 and 4) (Fig. 2). Thus, the amount of the phenylpropanoids accumulated in — P cells (6-d starvation) may be sufficient to protect cells from the peroxidation of lipids due to combined application of Al and Fe(II). Because the phenylpropanoids were not detected sig-

Antioxidant effect of phenylpropanoids

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Phenylpropanoids [O°320 nml Fig. 5 The inhibitory effect of the phenylpropanoid compounds extracted from — P cells on the Al-enhanced loss of viability in + P cells treated in nutrient medium that contained Fe(II). Phenylpropanoids were extracted from — P cells (6-d starvation). In the presence of various concentrations of the phenylpropanoid mixture, + P cells were treated with (•) or without (o) 150//M A1C13 in medium A (pH 4.0) containing 100 j/M FeSO4 for 18 h at a cell density of 100 mg fr wt/10 ml. After treatment, the cells (from 10ml aliquots) were collected and washed, and then viability was determined as described in Materials and Methods. 100% viability corresponds to the post-treatment growth of the control cells treated without both Al and phenylpropanoids. 1.0 OD32o corresponds to ca. 56/^M CA. All data show the means±S.E. (n=3).

nificantly in the medium which had been used for culturing cells for Pi starvation for 6 d (data not shown), it seems that the phenylpropanoids are not secreted from — P cells

Table 1

into medium. The localization of the phenylpropanoids inside cells has not been determined yet. Antioxidants against lipid peroxidation can act at different levels in the radical-chain reactions by (i) preventing first chain initiation by scavenging initiation radicals, (ii) chain breaking by scavenging intermediate radicals such as peroxyl and alkoxyl radicals to prevent continued hydrogen abstraction, (iii) chelating metal ions [e.g. Fe(II), Fe(III)] in forms that will not generate initiating radicals and/or will not decompose lipid peroxides to peroxyl or alkoxyl radicals, and so on (Halliwell and Gutteridge 1989). CA is known as a phenolic antioxidant of plant origin and is supposed to act as a chain-breaking antioxidant. Castelluccio et al. (1995) reported the antioxidant activities of the hydroxycinnamic acids in peroxidizing lipid systems mediated by metmyoglobin. The order of effectiveness is caffeic = chlorogenic>ferulic>p-coumaric acids, which probably depends on their chemical structures to scavenge alkoxyl and peroxyl radicals. Thus in the present experiment, it seems more likely that CA and the conjugated form of CA (peak 1 and CGA) work predominantly as a chain-breaking antioxidant. However, CA may also act as a metal chelator, because CA exhibited weak chelating activity of Al and Fe(II) (our unpublished results). Increase in PAL activity during P, starvation in tobacco cells—All phenylpropanoids are derived from cinnamic acid, which is formed from phenylalanine by the action of PAL. Thus, PAL is often speculated to be a key enzyme in phenylpropanoid metabolism. PAL can readily be induced by some environmental stresses (e.g., wounding, pathogen attack, UV) (for reviews, see Jones 1984, Ibrahim 1987, Lewis 1993, Dixon and Paiva 1995). Thus, we examined the

Inhibitory effects ot phenylpropanoid compounds on Al toxicity in + P cells

Al toxicity

Treatment

Phenylpropanoids

-Al

+ A1

Lipid peroxidation (nmol TBA-reactive substances/cells in 10-ml culture)

0.16 + 0.09 0.15 ±0.06

4.60± 0.86 0.39± 0.30

Al accumulation Gumol Al/cells in 10-ml culture)

0.07 ±0.04 N.D."

1.61 ± 0.17 O.38± 0.12

Fe accumulation Gumol Fe/cells in 10-ml culture)

0.07 ±0.03 0.10±0.03

0.34± 0.04 0.10± 0.03

Loss of viability (Viability, % of control)

100 112

±0 ±5

19 103

±7 ±11

° not determined. In the presence or absence of a mixture of phenylpropanoid compounds extracted from — P cells (6-d starvation) at a concentration of OD 320 1.0 (ca. 56 fiM CA equivalent), + P cells were treated with or without A1C13 (150^M) in medium A (pH 4.0) containing FeSO4 (100 jiM) for 18 h at a cell density of 100 mg fresh weight/10 ml. After treatment, the cells (from 10-ml aliquots) were collected and washed. Then the extent of lipid peroxidation, contents of Al and Fe and viability were determined as described in Materials and Methods. All data show the means±S.E. (n = 3).

Antioxidant effect of phenylpropanoids

955

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c 0.2 0.4 0.6 0.8 Protein cone, (mg/ml)

1.0 0 3 6 9 12 Duration of Pi starvation (days) Fig. 7 Changes in PAL activity and in phenylpropanoid content in cells during Pf starvation. + P cells were starved for P; in P r free modified MS medium for up to 12 d. At indicated times, the cells were withdrawn, and PAL activity (o) and the content of phenylpropanoids (•) were determined as described in Materials and Methods. The content of the phenylpropanoids in the extract was quantitated using the calibration coefficient of CA. All data show the mean±S.E. (n=3).

30

60

90

120

Time (min) Fig. 6 PAL activity in crude extracts prepared from + P cells and — P cells. Crude extracts were prepared from + P cells and — P cells (6-day starvation). The specific activity of PAL was determined in a protein dose-dependent (A) and a time-dependent (B) manner as described in Materials and Methods. All data show the means±S.E. (n = 3).

possibility that PAL activity might be induced during P; starvation in tobacco cells. Figure 6 indicates the specific activity of PAL in + P cells and — P cells (6-day starvation). — P cells exhibited 30 times higher specific activity of PAL than + P cells in both a protein-dependent and a time-dependent manner. During the culture without Pj, the specific activity of PAL increased quickly until 3 d after start of culture, reached a maximum value at 6 d, and then decreased until 12 d (Fig. 7). On the other hand, the amount of phenylpropanoids in cells increased gradually during the culture period reaching a maximum value at 12 d. These results indicate that an increase in phenylpropanoids in — P cells is based

Table 2 The effect of Al treatment on PAL activity in + P cells and - P cells Cells

+ p cells - p cells (6-d starvation) fold (-P/ + P)

PAL activity (nmolcinnamic acid (mg protein)"1 h~') -Al +A1 fold (+A1/-A1)

2.2±0. 1 57.0±O.8

3 .l±0.5

1.4

20 .0±0.4

0.4

25.9

6.5

+ P cells and —P cells (6-d starvation) were treated with or without A1CI3 (150 /iM) in medium A (pH 4.0) containing FeSO4 (100 fiM) for 18 h at a cell density of 100 mg fr wt/10 ml. After treatment, the cells were harvested for preparation of crude extracts. PAL activity in the extracts was determined as described in Materials and Methods. All data show the means±S.E. (n = 3).

Antioxidant effect of phenylpropanoids

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on an increase in the specific activity of PAL in — P cells. Knobloch and Berlin (1981) also reported that the enhanced formation of cinnamoyl putrescines during Ps depletion is preceded by an increase and subsequent decline of PAL activity in suspension cultured tobacco cells. In their system, — P cells exhibit a maximum value of PAL activity, which is 4 times higher than + P cells, at 2 d of culture in Pidepleted medium. By the treatment with Al in the presence of Fe(II), the specific activity of PAL was not significantly altered in + P cells, and rather decreased in — P cells (Table 2). In addition, contents of phenylpropanoids (peak 1,2, 3, and 4) were not affected by the treatment with Al and Fe(II) together in both + P cells and — P cells (data not shown). These results suggest that the phenylpropanoids accumulated in — P cells until a start of exposure to Al and Fe(II) play a role in the prevention of lipid peroxidation. The increase in PAL activity might be controlled by either transcriptional activation of the PAL gene or posttranslational activation of PAL (for review, Dixon and Paiva 1995). Molecular details of the regulation of PAL activity during Pj starvation are now under investigation in our laboratory. In summary, in cultured tobacco cells, — P cells are more tolerant to the peroxidation of lipids caused by the combination of Al and Fe than + P cells, and one of the tolerant mechanisms seems to be the accumulation of antioxidant molecules such as phenylpropanoid compounds (this paper) and /J-carotene (Yamamoto et al. 1996) in cells. Xie and Yokel (1996) reported that determinants of the Alfacilitated Fe-mediated peroxidation of lipids are phospholipid composition, pH and the concentrations of Al and Fe(II) in crude brain extracts and pure phospholipids. Thus quantitative and/or qualitative alterations in these determinants may be also possible tolerant mechanisms to the toxicity caused by a combination of Al and Fe in plant cells. The authors are grateful to Dr. N. Amrhein (Institute of Plant Sciences, Swiss Federal Institute of Technology, Switzerland) for his kind gift of AIP, JASCO International Co. Ltd. for providing the MS spectra and Dr. T. Yoshida (Okayama Univ., Japan) for his useful suggestions about antioxidant effects of phenolic compounds. This work was supported in part by a Grant-in-Aid for General Scientific Research (no. 08640829) from the Ministry of Education, Science, Sports and Culture of Japan, the Ohara Foundation for Agricultural Science, Hayashi Memorial Foundation for Female Natural Scientists, and Showa Shell Sekiyu Foundation for the Promotion of Environmental Research.

References Castelluccio, C , Paganga, G., Melikian, N., Bolwell, G.P., Pridham, J., Sampson, J. and Rice-Evans, C. (1995) Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBSLett. 368:

188-192. Dixon, R.A. and Paiva, N.L. (1995) Stress-induced phenylpropanoid metabolism. Plant Cell!: 1085-1097. Edwards, R. and Kessmann, H. (1992) Isoflavonoid phytoalexins and their biosynthetic enzymes. In Molecular Plant Phathology, A Practical Approach. Vol. II. Edited by Gurr, S.J., McPherson, M.J. and Bowles, D J . pp. 45-62. IRL Press at Oxford Univ. Press Inc., New York. Gutteridge, J.M.C., Quinlan, G.J., Clark, I. and Halliwell, B. (1985) Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts. Biochim. Biophys. Ada 835: 441-447. Halliwell, B. and Gutteridge, J.M.C. (1989) Free Radicals in Biology and Medicine. 2nd Edition, Clarendon Press, Oxford. Horst, W.J. (1995) The role of the apoplast in aluminium toxicity and resistance of higher plants: a review. Z. Pflanzenernahr. Bodenk. 158: 419-428. Ibrahim, R.K. (1987) Regulation of synthesis of phenolics. In Cell Culture and Somatic Cell Genetics of Plants. Edited by Constabel, F. and Vasil, I.K. Vol. 4. pp. 77-95. Academic Press, Inc., U.S.A. Jones, D.H. (1984) Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23: 1349-1359. Knobloch, K.-H. and Berlin, J. (1981) Phosphate mediated regulation of cinnamoyl putrescine biosynthesis in cell suspension cultures of Nicotiana tabacum. Planta Med. 42: 167-172. Kochian, L. V. (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annu. Rev. Plant Physiol. Mol. Biol. 46: 237-260. Lewis, N.G. (1993) Plant phenolics. In Antioxidants in Higher Plants. Edited by Alscher, R.G. and Hess, J.L. pp. 135-169. CRC Press, Baca Raton. Mizusaki, S., Tanabe, Y., Noguchi, M. and Tamaki, E. (1971) pCoumaroylputrescine, caffeoylputrescine and furuloylputrescine from callus tissue culture of Nicotiana tabacum. Phytochemistry 10: 13471350. Nakamura, C , Telgen, H.V., Mennes, A.M., Ono, H. and Libbenga, K.R. (1988) Correlation between auxin resistance and the lack of a membrane-bound auxin binding protein and a root-specific peroxidase in Nicotiana tabacum. Plant Physiol. 88: 845-849. Ono, K., Yamamoto, Y., Hachiya, A. and Matsumoto, H. (1995) Synergistic inhibition of growth by Al and iron of tobacco (Nicotiana tabacum L.) cells in suspension culture. Plant Cell Physiol. 36: 115-125. Oteiza, P.I. (1994) A mechanism for the stimulatory effect of aluminum on iron-induced lipid peroxidation. Arch. Biochem. Biophys. 308: 374-379. Rengel, Z. (1992) Role of calcium in aluminium toxicity. New Phytol. 121: 499-513. Rengel, Z. (1996) Uptake of aluminium by plant cells. New Phytol. 134: 389-406. Shinozaki, M., Asada, K. and Takimoto, A. (1988) Correlation between chlorogenic acid content in cotyledones and flowering in Pharbitis seedlings under poor nutrition. Plant Cell Physiol. 29: 605-609. Taylor, G.J. (1988) The physiology of aluminum phytotoxicity. In Metal Ions in Biological Systems. Edited by Sigel, A.H. and Sigel, A. Vol. 24. pp. 165-198. Marcel Dekker, New York. Verstraeten, S.V., Nogueira, L.V., Schreier, S. and Oteiza, P.I. (1997) Effect of trivalent metal ions on phase separation and membrane lipid packing: role in lipid peroxidation. Arch. Biochem. Biophys. 338: 121127. Xie, C.X. and Yokel, R.A. (1996) Aluminum facilitation of iron-mediated lipid peroxidation is dependent on substrate, pH, and aluminum and iron concentrations. Arch Biochem. Biophys. ill: 222-226. Yamamoto, Y., Hachiya, A. and Matsumoto, H. (1997) Oxidative damage to membranes by a combination of aluminum and iron in suspension-cultured tobacco cells. Plant Cell Physiol. 38: 1333-1339. Yamamoto, Y., Masamoto, K., Rikiishi, S., Hachiya, A., Yamaguchi, Y. and Matsumoto, H. (1996) Aluminum tolerance acquired during phosphate starvation in cultured tobacco cells. Plant Physiol. 112: 217-227. Yamamoto, Y., Ono, K. and Matsumoto, H. (1995) Determining factors for aluminium toxicity in cultured tobacco cells: medium components and cellular growth conditions. In Plant-Soil Interactions at Low pH: Principles and Management. Edited by Date, R.A., Grundon, N. J., Rayment, G.E. and Probert, M.E. pp. 359-361. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Antioxidant effect of phenylpropanoids Yamamoto, Y., Rikiishi, S., Chang, Y-C, Ono, K., Kasai, M. and Matsumoto, H. (1994) Quantitative estimation of Al toxicity in cultured tobacco cells: correlation between Al uptake and growth inhibition. Plant Cell Physiot. 35: 575-583.

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Zoii, J. and Amrhein, N. (1992) Inhibitors of phenylalanine ammmonialyase: 2-aminoindan-2-phosphonic acid and related compounds. Liebigs Ann. Chem. 625-628. (Received May 18, 1998; Accepted July 1, 1998)