interna1 detoxification mechanism of ai in hydrangea' · 100 ~lm cac1, solution at ph 4.5: (a)...

Plant Physiol. (1 997) 11 3: 1033-1 039

Interna1 Detoxification Mechanism of AI in Hydrangea'

ldentification of AI Form in the Leaves

Jian Feng Ma*, Syuntaro Hiradate, Kyosuke Nomoto, Takasi Iwashita, and Hideaki Matsumoto

Research lnstitute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 71 O, Japan (J.F.M., H.M.); Division of Plant Ecology, National lnstitute of Agroenvironmental Sciences, Tsukuba Norin Danchi, P.O. Box 2,

Tsukuba, lbaraki 305, Japan (S.H.); and Suntory lnstitute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 61 8, Japan (K.N., T.I.)

An internal detoxification mechanism for AI was investigated in an AI-accumulating plant, hydrangea (Hydrangea macrophylla), focusing on AI forms present in the cells. l h e leaves of hydrangea contained as much as 15.7 mmol AI kg-' fresh weight, and more than two-thirds of the AI was found in the cell sap. Using "AI- nuclear magnetic resonance, the dominant peak of AI was observed at a chemical shift of 11 to 12 parts per million in both intact leaves and the extracted cell sap, which is in good accordance with the chemical shift for the 1 :1 AI-citrate complex. Purification of cell sap by molecular sieve chromatography (Sephadex C-1 O) combined with ion-exclusion chromatography indicated that AI in fractions with the same retention time as citric acid contributed to the observed "AI peak,in the intact leaves. l h e molar ratio of AI to citric acid in the crude and purified cell sap approximated 1. The structure of the ligand chelated with AI was identified to be citric acid. Bioassay experiments showed that the purified AI complex from the cell sap did not inhibit root elongation of corn (Zea mays L.) and the viability of cells on the root tip surface was also not affected. lhese observations indicate that AI i s bound to citric acid in the cells of hydrangea leaves.

A1 toxicity is primarily characterized by the inhibition of root elongation, with no appearance of clearly identifiable symptoms in plant tops. This is because A13+, a toxic ionic species, has a high binding ability with cellular components of roots, and usually shows little translocation to the upper parts of plants. Most plants contain not more than 0.2 mg AI 8-l dry weight. However, some plants, known as "A1 accumulators," may contain more than 10 times this leve1 of A1 without any AI injury. Tea plants are typical AI accumulators; the A1 content in these plants can reach as high as 30 mg 8-l dry weight in old leaves, although

This study was supported in part by Grants-in-Aid for Encour- agement of Young Scientists from the Ministry of Education, Sci- ence, Sports and Culture of Japan, by Sunbor, and by the Ohara Foundation for Agricultura1 Research.

* Corresponding author; e-mail majQrib.okayama-u.ac.jp; fax 81-86-421-0699.

103

young leaves contain only around 0.6 mg A1 8-l dry weight (Matsumoto et al., 1976). Some trees in the tropical cloud forest, such as Richeria grandis, have been reported to accumulate more than 1 mg g-* in their leaves (Cuenca et al., 1990). Hydrangea (Hydrangea macrophylla) is also a well known Al-accumulating plant, and the relationship between A1 and the blue coloration of hydrangea sepals has been thoroughly investigated (Takeda et al., 1985a, 1985b). In contrast to tea plants, which usually accumulate AI over long periods (more than 1 year), hydrangea plants can accumulate as much as 5 mg A1 8-l dry weight in the leaves within severa1 months (J.F. Ma, unpublished data).

Recent research indicated that AI can enter the symplasm of root cells fairly quickly, and A1 phytotoxicity might be the result of a symplasmic A1 interaction (Lazof et al., 1994; Vitorello and Haug, 1996). Symplasmic solutions usually have a pH above 7.0. Although the concentration of free AI3+ is decreased to less than 1O-I' M at pH 7.0 due to the formation of insoluble Al(OH),, such low concentrations are still potentially phytotoxic because of the strong affinity of A1 for oxygen donor compounds such as inorganic phosphate, ATP, RNA, DNA, proteins, carboxylic acids, and phospholipids (Martin, 1988). These observations led us to suggest that internal detoxification mechanisms are re- quired for tolerance to high A1 in Al-accumulating plants. However, the mechanisms of AI detoxification in these plants are still poorly understood.

Severa1 potential mechanisms of tolerance after A1 has entered the symplasm have been addressed (Taylor, 1991; Kochian, 1995). These include chelation in the cytosol, com- partmentalization in vacuoles, AI-binding proteins, evolu- tion of Al-tolerant enzymes, and elevated enzyme activity. However, experimental data supporting these hypotheses have yet to be reported. In the present study, the internal detoxification mechanism of AI was investigated in hydrangea, focusing on the identification of A1 forms present in the leaves.

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1034 Ma et al. Plant Physiol. Vol. 1 1 3, 1997

MATERIALS AND METHODS

Hydroponic Culture

To confirm the role of A1 in the blueing of hydrangea (Hydrangea macrophylla) sepals, plants were cultured hydroponically with or without Al. Hydrangea plants with blue sepals were purchased at a local market. Stems with one or two leaves were cut and the cuttings were grown in ver- miculite. After a 4-week growth period, the rooted cuttings were transplanted into 1-L plastic pots (two per pot) with continuously aerated nutrient solution in a greenhouse; one-tenth-strength Hoagland solution was used for the culture. After another 2 weeks, the plants were given nutrient solution with (100 p~ Al) or without A1 (three rep- licates for each condition). The A1 solution was prepared from AlC1,.6H2O (Wako, Tokyo, Japan). The solution was adjusted to pH 4.0 with 1 N HCl and replaced every week. The culture period was from May 1995 to May 1996.

Extraction and Purification of the Cell Sap

For the extraction of cell sap, detached leaves were washed with distilled water and stored at -80°C until use. Before use, frozen samples were ground by hand and then placed on filters in centrifuge tubes (Centricut U-50, molecular weight cutoff 50,000, Biofield, Tokyo, Japan). After thawing at room temperature, the samples were centri- fuged at 10,OOOg for 20 min to obtain the cell sap. The pH of the cell sap was measured using a pH meter (model 8-212, Horiba, Kyoto, Japan), and the concentrations of A1 and organic acids were determined immediately by the methods described below.

For purification of the A1 complex, 4 mL of freshly prepared cell sap was first applied to a 16 mm X 170 cm column of Sephadex G-10 and fractionated (3 mL each, nos. 1-65) in a cold room (4°C). Dilute perchloric acid solution (pH 4.6, the same pH as the cell sap) was used as the eluant, and passed with a peristaltic pump at a flow rate of 0.72 mL min-l. The concentrations of A1 and organic acids in each fraction were determined using the methods described below. Fractions 29 to 39, which contained both A1 and organic acids, with a retention time of 8.8 min in HPLC chromatography, were concentrated at 40°C using a rotary evaporator (Eyela, Tokyo, Japan). The concentrated fraction was applied again to the Sephadex G-10 column as described above. Fractions 28 to 38 containing the A1 complex were concentrated and then subjected to 27A1-NMR measurement, as described below.

Further purification of the ligand in the A1 complex was achieved by passing the cell sap (purified as described above) through a cation-exchange column (Amberlite IR- 120, H+ form, 16 mm X 14 cm (Organo, Tokyo, Japan), followed by passage through an anion-exchange column (AG 1x8, 100-200 mesh, formate form, 16 mm X 14 cm, (Bio-Rad). The anionic fractions were eluted using 6 N

formic acid and concentrated. Final purification was per- formed by injecting the fraction onto an ion-exclusion column (Shimpack SCR-l02H, Shimadzu, Kyoto, Japan) and eluted with dilute perchloric acid solution (pH 2.1) at a flow rate of 0.8 mL min-l. The chromatographic fractions

were monitored at 220 nm, and those with a retention time of 8.8 min were collected and evaporated to dryness. 'H- NMR spectra of purified fractions (in 'H,O) were recorded on a 500-MHz spectrometer (DMX500, Bruker, Karlsruhe, Germany). Mass spectra of purified fractions were measured on a spectrometer (JMX-HX110, JEOL) using fast- atom bombardment negative ionization with dithiodietha- no1 matrix.

"AI-NMR Measurement

27A1-NMR spectra were obtained at 156.3 MHz (JNM- a600 spectrometer, JEOL). Detached leaves .were cut with scissors and then placed in NMR tubes 10 mm in diameter. Solution samples were analyzed using NMR tubes 5 mm in diameter. The parameters used were: frequency range, 62.5 kHz; data point, 33,000 (131,000 for intact leaves); acquisi- tion time, 0.24 s (0.5 s for intact leaves); and 5,000 scans (4,098 for intact leaves). AlC1, (0.2 mM AlC1, in 0.1 N HC1) was used as an externa1 reference for calibration of the chemical shift (O ppm). The 27A1-NMR spectrum of the Al-citrate complex, prepared by mixing equimolar AlCl, and citric acid, was also recorded for comparison. The pH of the mixture was adjusted to 4.6 with 0.2 N NaOH and allowed to stand for 2 d before passing through a Sephadex G-10 column, as described above.

Determination of AI and Organic Acids

A1 was determined by graphite furnace atomic absorp- tion spectrophotometry (Hitachi 2-9000, Tokyo, Japan) di- rectly or after digestion. To analyze organic acids, HPLC was employed using an ion-exclusion column (Shimpack SCR-l02H, 8.0 mm X 30 cm, Shimadzu) with a guard column (6.0 mm x 5 cm). The mobile phase was dilute perchloric acid solution (pH 2.1) run at 40"C, and peaks were detected at 425 nm after reaction with 0.2 mM bromthymol blue, 15 mM NaH,PO,, 2 mM NaOH in 5% metha- nol. Some samples were detected by UV A220 with no reactive reagent. The flow rate of the mobile phase was 0.8 mL min-' and that of the reactive phase was 0.6 mL min-l.

Bioassay of AI Toxicity

The toxicity of A1 in the cell sap of hydrangea leaves was tested by investigating inhibition of root elongation in corn (Zea mays L. cv Golden Cross Bantam). Corn seeds were soaked in water for 10 h and then germinated on moist filter paper in a 30°C incubator. After 2 d, the seedlings were transplanted into a tray containing 100 p~ CaC1, solution at pH 4.5 in a growth chamber under the following conditions: 27°C day and 20°C night, 60% RH, light inten- sity of 40 W m-', and a 14-h photoperiod. The solution was renewed every day. After another 2 d, seedlings of similar size were selected and subjected to various treatments in 100 ~ L M CaC1, solution at pH 4.5: (a) -A1 (control, no A1 addition); (b) +A1 (addition of 20 PM AlCl,); or (c) +sap (purified cell sap containing 20 p~ A1 from the hydrangea leaves as described above). The treatment period was 22 h. Root length was measured with a ruler before and after treatment. After treatment, the roots were placed in dis-

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Interna1 Detoxification Mechanism of AI 1035

tilled water for 5 min and then stained using 0.1% eriochrome cyanine R solution for 15 min or 3 mg L-' propidium iodide solution for 15 min. After washing, the roots stained with eriochrome cyanine R were observed with a light microscope (model B061, Olympus), and those stained with propidium iodide were examined with a microscope photometer (model MPM-800, Zeiss) under epifluorescence illumination, a band-pass 540- to 552-nm exciter filter, and a long-pass 590-nm barrier filter. A plan-neofluar lens (5X / 0.15) was used to observe red-fluorescing cells.

RESULTS

It is well known that the color of hydrangea sepals changes from red to blue when soil pH is shifted from weak alkaline or neutra1 to acidic. However, it was found that it is the A1 dissolved in acid soils that is responsible for the blue color of the sepals, not soil pH itself (Allen, 1943). To confirm the effect of A1 on hydrangea, plants were cultured hydroponically in the presence or absence of Al. There were no visible differences in growth between the plants treated with and without Al. However, the sepals of plants grown in the solution containing A1 were blue, whereas those of plants grown at the same solution pH but without A1 were pink.

The hydrangea leaves with blue sepals contained as much as 15.7 mmol A1 kg-' fresh weight (Table I), whereas those with pink sepals contained only 0.23 mmol A1 kg-'

gea leaves with blue sepals was extracted in the cell sap, which had a pH of 4.6 (Table I).

Because 27A1-NMR spectroscopy is the only definitive means of identifying A1 species (Karlik et al., 1982, 1983a), this method was used to study the A1 forms accumulating in hydrangea leaves. Three peaks were observed in the intact leaves, but the dominant peak appeared at a chemical shift of 12.4 ppm (Fig. lA), suggesting that A1 in the leaves was mainly in a hexacoordinated complex (Haragu- chi and Fujiwara, 1969). No signals corresponding to Al(H20)3+ (O ppm), A1-F (-1 ppm), Al-polyphosphate (-3-7 ppm), or Al(OH),- (+80 ppm) complexes were observed. The chemical shift of the extracted cell sap was at 11.3 ppm (Fig. lB), which is consistent with that of intact leaves (Fig. lA), suggesting that the A1 form did not change after it was extracted in the cell sap. The slight difference in the chemical shift between the intact leaves and the cell sap might have been the result of broadening of the peaks because the 27Al nucleus is quadrupolar (spin 5/21.

To identify the ligands in the A1 complex, the cell sap was purified by molecular sieve chromatography using

fresh weight. More than two-thirds of the A1 in the hydran-

Table 1. AI concentrations in the leaves and cell sap of H. macrophylla

mmol kg-' fresh wt % mM

15.66 87.7 13.70 4.6 76.7

I I I I I I 20 15 10 5 o -5

Chemical shift (ppm)

Figure 1. *'AI-NMR spectra of intact hydrangea leaves (A), cell sap

(B), cell sap purified by a second round of Sephadex G-10 column chromatography (C), and 1 :I AI-citrate complex (D). Spectra were measured at 156.3 MHz. See "Materiais and Methods" for measurement conditions.

Sephadex G-10 to avoid the separation of A1 from the chelated ligands. Since A1 forms stable complexes with oxygen donor ligands, the chelators in biological systems may be carboxylate and phosphate groups, inorganic phosphate, nucleotides, and polynucleotides (Martin, 1988). Based on the observed chemical shift in the 27A1-NMR spectrum both in the intact leaves and the cell sap (Fig. 1, A and B), it was likely that A1 was chelated with organic acids (at the chemical shifts between O and +50 ppm), so the concentrations of AI and organic acids in each fraction eluted from Sephadex G-10 were monitored. In the present study, organic acids were detected using HPLC. The samples were first eluted from the ion-exclusion column, followed by reaction with bromthymol blue, and detection at 425 nm. Severa1 peaks were detected in the cell sap (Fig. 2A). By passing the cell sap through a Sephadex G-10 column, a peak at retention time 8.8 min was first eluted, followed by a mixture of other peaks (Fig. 2B). When fractions 29 to 39, mainly containing the peak at retention time 8.8 min, were concentrated, about 80% of the A1 found in the crude cell sap was found (Table 11). The concentrated fraction was applied to the Sephadex G-10 column once more, and fractions 28 to 38, only containing the peak at 8.8 min, were concentrated (Fig. 2C). These fractions contained about 65% of the A1 found in the crude cell sap (Table 11). The chemical shift of the cell sap purified by the second



1036 Ma et al. Plant Physiol. Vol. 11 3, 1997

L I , 1 1 I I O 5 10 O 5 10 15

Retention time (min) Retention time (min)

Figure 2. HPLC profile of ligand in the AI complex in the crude and purified cell sap on an ion-exclusion column. A, Cell sap of hydrangea leaves; B, cell sap purified by the first round of Sephadex G-10 column chromatography; C, cell sap purified by the second round of Sephadex G-1 O column chromatography; D, ligand purified by ion- exclusion column chromatography, E, citric acid; F, purified ligand by ion-exclusion column; G, citric acid. A to E, Detected at 425 nm; F and G, detected at 220 nm.

round of Sephadex G-10 chromatography was observed at -10.9 ppm in the 27A1-NMR spectrum (Fig. 1C). The consis- tency of chemical shifts observed in the purified cell sap and intact leaves suggests that the fraction with a retention time of 8.8 min was attributable to the ligand chelated with A1 in the hydrangea leaves. To identify this peak, comparison of the retention time was made with standards of organic acids, and citric acid was found to have the same retention time (8.8 min) (Fig. 2E). The chemical shift of the prepared complex of Al-citrate (1:l) was observed at 10.7 ppm in the 27Al-NMR spectrum (Fig. 1D). These results indicated that A1 was bound to citric acid in hydrangea leaves, and citric acid in the crude and purified cell sap was therefore quantified. The molar ratio of A1 to citric acid approximated 1 in each case (Table 11). Sephadex G-10 chromatography also indicated the ratio to be about 1 in

Table II. Amounts of AI and citric acid in tbe crude and purified cell sap of H. macropbylla

Cell sap was purified twice by Sephadex (3-10 column chroma- tonraDhv usinp. dilute Derchloric acid solution b H 4.6) as the eluant.

Sample

~ ~ ~~

Citric AI/Citric Acid Acid

AI

pmol

Cell sap 54.80 44.48 1.23 Sephadex G-1 O

First (fractions 29-39) 44.24 33.77 1.31 Second (fractions 28-38) 35.1 8 30.38 1.16

2500

9 2000

1 a v

3 1500

.3 d c 8 1000 B 8

500

O

AI ........o........ Cinic acid

20 40 60

Fraction number

2500

2 2000 a 9 B

1500 .o B

v

.- +

1000 8 2

8

'a

500 f V

O

Figure 3. Molecular-sieve chromatograms (first Sephadex G-1 O) of the cell sap of hydrangea leaves. Dilute perchloric acid solution (pH 4.6) was used as the eluant, and fractions (3 mL each) were collected at a flow rate of 0.72 mL min-'.

each fraction (Fig. 3). These results demonstrated that A1 is chelated with citric acid at a 1:l ratio.

To further identify the structure of the AI-binding ligand, A1 was separated from the complex by passing the purified cell sap through cation- and anion-exchange columns. Final purification of the ligand by ion-exclusion column HPLC yielded a single peak, which had the same retention time as citric acid with detection at either 425 or 220 nm (Fig. 2, D-G). The 'H-NMR spectrum of purified ligand displayed signals corresponding to methylene protons at 2.74 ppm (lH, doublet, = 15.8 Hz) and 2.92 ppm (lH, doublet, J = 15.8 Hz) (Fig. 4). Both the chemical shifts and the coupling constants were consistent with those of citric acid. The negative-ion fast-atom bombardment mass spectrum of the purified fraction exhibited the pseudomolecular ion peaks [M-H]- at m / z 191. These results further demonstrated that the ligand chelated with A1 in hydrangea leaves was citric acid.

Because inhibition of root elongation is the primary response to A1 toxicity, phytotoxicity of the AI complex purified from the cell sap was examined by investigating the effect on root elongation in corn. Addition of 20 p~ A13+ induced 50% inhibition of root elongation within a period of 22 h, whereas the addition of the A1 complex at the same A1 concentration did not show any inhibitory effect on root elongation (Fig. 5). Eriochrome cyanine R staining, a method to asses A1 toxicity visually (Aniol, 1995), showed strong staining in the apex in roots treated with AI3+, whereas those treated with the A1 complex showed no staining (data not shown). Cell viability on the root tip surface was observed using propidium iodide staining (Jones and Senft, 1985). Propidium iodide can pass through damaged cell membranes and intercalates with DNA and RNA to form a bright-red fluorescent complex seen in the nuclei of dead cells. Since propidium iodide is excluded by intact cell membranes, it is an effective method to identify nonviable cells. The cell viability was decreased by the addition of A13', but not by the addition of the Al complex (Fig. 6). These results indicated that the A1 complex puri-




4 3 2 1

Chemical shift (ppm) Figure 4. 'H-NMR spectrum of purified ligand chelated with AI in the hydrangea leaves. Spectrum was recorded at 500 MHz in 'H,O solution.

fied from the cell sap of hydrangea leaves was nonphytotoxic.

D I SC U SS I ON

The anthocyanin in both red and blue sepals of hydrangea has been identified as delphinidin 3-monoglucoside (Takeda et al., 198513). The blue color of hydrangea sepals is thought to be due to the formation of a blue complex containing the anthocyanin, Al, and a co-pigment (delphinidin 3-glucoside-aluminum-3-caffeoylquinic acid) (Takeda et al., 1985a). A1 may play a role in stabilizing the interaction between the quinic ester and the anthocyanin. In the present study, the role of A1 in the blueing of the sepals was confirmed in hydrangea plants cultured hydroponically with or without Al.

In hydrangea plants with blue sepals more A1 is accumulated in the leaves. However, the interna1 detoxification mechanisms of A1 in the leaves have yet to be elucidated. In the present study, we focused on identification of A1 forms present in hydrangea leaves. A1 was shown by the following results to be bound to citric acid in the leaves: (a) the chemical shift of 27Al in the intact leaves was consistent with that of the Al-citrate complex (1:l) (Fig. 1); (b) the retention time of the ligand chelated with A1 was the same as that of citric acid on HPLC (Fig. 2); (c) the molar ratio of A1 to citric acid in both crude and purified cell sap approximated 1 (Table 11; Fig. 3); (d) molecular weight determination with fast-atom bombardment MS and the 'H chemical shifts and coupling constants of purified ligand coincided with those of citric acid (Fig. 4); and (e) the A1 complex contained in the leaves was nonphytotoxic (Figs. 5 and 6). Recently, A1 in the soluble fraction of wheat roots

was reported to be present in the form of the Al-citrate complex (Jones and Kochian, 1995).

Previous studies suggested that A1 was bound to F in tea plants, because A1 concentration showed a good correla- tion with that of F (Yamada and Hattori, 1977, 1980). How- ever, peaks corresponding to AI-F complexes (chemical shift of about -1 ppm) in tea leaves were not observed in the 27A1-NMR spectrum, whereas a peak at a chemical shift of 16 to 20 ppm was observed (Nagata et al., 1992). The authors of that study proposed that most of the A1 was bound to catechins, which are also very abundant in tea plants. The A1-F complex was later suggested to be in- volved in uptake and translocation of A1 (Nagata et al., 1993). A previous study showed that A1 was rapidly taken up and accumulated into polyphosphate complexes in the vacuoles of the mycorrhizal basidiomycete Laccaria bicolor (Martin et al., 1994). In the present study, signals corresponding to neither the A1-F complex nor to the catechins or polyphosphate complexes were observed (Fig. 1).

Citric acid has four potential donor sites, one central and two terminal carboxyl groups, and an alcoholic hydroxyl group on the central carbon atom. However, the coordina- tion model of the Al-citrate complex is still not definitive (Ohman, 1988). From the observed chemical shifts of 27Al (about 11-12 ppm), the most likely structure in an equimolar A1:citric acid solution has A1 bound to citrate via two carboxyls and one hydroxyl, producing two six-membered rings, although other models have also been proposed (Karlik et al., 1983b; Motekaitis and Martell, 1984; Feng et al., 1990).

The standard stability constant of Al-citrate complex was reported to be 8.1 (Martin, 1988). However, the conditional stability constant becomes 11.7 and 12.4 at pH 7.0 and 7.4, respectively, which are significantly stronger than that for the A1-ATP complex (10.9). This strong chelation capacity

3'01

d 1.5

0 1.0 i-'

0.5

0.0

T I

I

-Al 1 +A1 3+ I +Sap

Treatment Figure 5. Effects of different AI species on root elongation in corn (cv Golden Cross Bantam). Roots were treated with 20 FM AI3+ (+AI3') or 20 p~ AI complex (+Sap) purified from the cell sap of hydrangea leaves for 22 h.



1038 Ma et al. Plant Physiol. Vol. 113, 1997

-Al

+A1

+Sap

1 mmFigure 6. Cell viability of root tip surface (1st cm of root) detected by propidium iodide staining in corn (cv Golden CrossBantam). Roots were treated for 22 h in 100 JAM CaCI2 solution at pH 4.5 containing 0 IJ.M Al (-AI), 20 JJ.M AI3+ ( + AI), and20 JIM Al complex (+Sap) purified from the cell sap of hydrangea leaves. Bright-red fluorescence is indicative of dead cells.For details, see "Materials and Methods."

could effectively reduce the activity of Al in the cytosol atpH above 7.0 and prevent formation of the complex be-tween Al and cellular components such as ATP and DNAand would, therefore, decrease Al phytotoxic effects. Invitro studies indicated that citric acid reduced the Al-induced inhibition of a K+-stimulated, Mg2+-dependentplasma membrane ATPase activity in Pisum sativum or Z.mays (Matsumoto and Yamaya, 1986; Suhayda and Haug,1986). It was also demonstrated that application of citricacid can partially restore the Al-induced loss of structure incalmodulin once an Al-calmodulin complex had beenformed, or, if added prior to Al addition, protect the reg-ulatory protein from undergoing a loss of a-helix content(Suhayda and Haug, 1984, 1986). The present study alsoconfirmed that the Al-citrate complex is nonphytotoxicfrom its lack of an effect on root elongation of corn. Re-cently, secretion of citric acid has also been suggested as aexclusion mechanism of Al tolerance in snapbean (Mi-yasaka et al., 1991) and corn (Pellet et al., 1995).

In conclusion, the internal detoxification of Al isachieved by the formation of a strong Al-citrate complex, anontoxic form of Al, in hydrangea leaves. However, whichforms of Al are taken up by the roots and translocated intothe upper parts of the plants remain to be elucidated. Thelocalization of the Al-citrate complex in hydrangea leaves,that is, whether the complex is present in the cytosol or invacuoles, also needs to be addressed in future studies. Weattempted to determine the localization of the Al complexin cells by analyzing the chemical shift of 27A1 based on thesupposition that it may change at different pH values in amanner similar to phosphate. However, the chemical shiftswere unaffected at any pH values between 4.5 and 7.5.Isolation of protoplasts and vacuoles is currently inprogress in our laboratory.

Received September 23, 1996; accepted January 10, 1997.Copyright Clearance Center: 0032-0889/97/113/1033/07.

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interna1 detoxification mechanism of ai in hydrangea' · 100 ~lm cac1, solution at ph 4.5: (a)...

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