chapter 46 - plant roots under aluminum stress - toxicity and tolerance

8/4/2019 Chapter 46 - Plant Roots Under Aluminum Stress - Toxicity and Tolerance

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46

Plant Roots Under Aluminum Stress: Toxicity and Tolerance

Hideaki MatsumotoOkayama University, Okayama, Japan

I. DISTRIBUTION OF ACID SOILS AND

THEIR NATURE

Acid soils occupy 3.95 billion ha (30%) of the worlds

ice-free land area (Baligar et al., 1998), comprising

both the tropical and temperate belts. The distribution

of acid soil in selected regions in the world is shown in

soil acidity, acid soil can be classified into 10 groups.

Oxisols and Ultisols are the major acid soils in the

tropical region and occupy 22% (846 m ha) and 18%

(727 m ha), respectively, of the acid soil area in theworld. Inceptisols including the sulfate soils of many

tropical river deltas are very acidic owing to acid

formed upon oxidation of sulfides. The level of acid-

ification of acid soil generally reflects the degree of

weathering and leaching it has experienced (Baligar

et al., 1998). In addition to the natural factors that

affect weathering, agricultural farming processes such

as the excessive supply of inorganic fertilizers or

removal of cations by harvest lower the pH.

Furthermore, the acidity of soils is gradually increased

owing to environmental pollution and acid rain. Acid

soils are infertile because they lack the basic nutrients,

such as Ca2, Mg2, and K. Acid soils are character-

ized by high content of toxic elements such as Al, Mn,

and Fe or deficiency of Ca2, Mg2, K, N, and P.

Most acid soils have low cation exchange capacity,

leading to loss of essential minerals and to poor crop

production.

II. ALUMINUM TOXICITY IN ACID SOILS

A. Occurrence and Chemistry of Al

Exchangeable Al and Mn are the major toxic elements

in most acid soils. In most Oxisols and Ultisols, Al

occupies 494% of the cation exchange sites (Baligar

et al., 1998). Al is the most abundant metal in the

earths crust.

Aluminum exists in the soil in insoluble aluminosi-

licates or oxides. It has complicated chemical form and

biological function. At pH < 5, Al3 exists as the octa-

hedral hexahydrate, AlH2O36 , often abbreviated as

Al3. As the solution becomes less acidic, AlH2O36

undergoes successive deprotonations to yield

AlOH2 and AlOH2 . In neutral solution AlOH3precipitates as gibbsite which redissolves in basic solu-

tions owing to formation of tetrahedral AlOH4 as

aluminate anion. Time-dependent formation of poly-

nuclear species may also take place (Martin, 1986).

Since Al toxicity differs with the chemical form of Al,

many studies have been done regarding this interac-

tion, especially between Al3 and mononuclear

hydroxy-Al. Generally Al3

is more phytotoxic thanAlOH2 or AlOH2 , but Alva et al. (1986) and

Kinraide and Parker (1987) reported that dicotyledo-

nous plants may be more sensitive to AlOH2 and

AlOH2 than to Al3. The difference in behavior of Al

species between monocots and dicots may be related to

the fact that dicots have a much higher CEC in their

821

Copyright 2002 by Marcel Dekker, Inc.

Table 1. Depending on the degree of weathering and


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cell wall than monocots, but further studies are needed

to elucidate the cause.

In the soil solution Al3 reacts not only with OH

but also with phosphate, F, SO24 , silicate, and a large

number of organic ligands. Under specific conditions

of OH/Al ratio, total Al, and stirring rate,AlO4Al12OH24H2O712 (Al13 polymer), which is

highly toxic, can be formed (Parker et al., 1989).

Nevertheless, Al13 was not observed in soil solutions

(Funakawa et al., 1993). Rhizotoxicity of the alumi-

nate ion, AlOH4 , which is formed at an alkaline

pH was tested with wheat and red clover. Root elonga-

tion was inhibited to < 4% by 25M AlOH4 at pH 8

but not at pH 8.9, where elongation was unaffected.

Thus, AlOH4 is nontoxic, and the inhibition at a

lower pH is attributable to the Al13 formed

(Kinraide, 1990).

B. Inhibition of Root Elongation by Al

Inhibition of root elongation is the first visible symp-

tom of Al stress. In most plant species, root elongation

is markedly inhibited by Al at the mol level in a

simple solution containing Ca2 alone. Inhibition of

root elongation of Al-sensitive maize occurred within

30 min of Al treatment (Llungany et al., 1995).

However, inhibition of root elongation is reduced in

the presence of other ions, because the interaction with

other coexisting ions reduces the toxicity of Al3.

Changes in the electric charge of the root surface by

other ions, especially cations, affect the accessibility of

Al3. Root elongation in Al-sensitive wheat cultivar,

Scout 66, was apparently inhibited by a 3-h treatment

with 5M Al, but that of Atlas 66 was inhibited to the

same degree only by a 10-fold higher concentration of

elongation zone) accumulated more Al and plays a

major role in the Al perception mechanism.

Indeed, only the apical 23 mm of maize and pea

roots need to be exposed to Al for the inhibition of

root elongation to take place (Delhaize and Ryan,

1995; Matsumoto et al., 1996). In near-isogenic

wheat (Triticum aestivum) lines differing in Al toler-

ance, root apices of Al-sensitive genotypes were stainedwith hematoxylin after a short exposure to Al (10 min

h). Apices of Al-tolerant seedlings showed less inten-

sive staining (Delhaize et al., 1993a). This indicates

that inhibition of root elongation by Al varies among

plant species or cultivars. Similar results were obtained

with Al-tolerant wheat (Atlas 66) and Al-sensitive

wheat (Scout 66) exposed to Al for 1 day (Sasaki et

differences in Al accumulation in the root apex are

related to differences in Al sensitivity, (2) inhibition

of root growth is related to the Al content in the

root apex, and (3) tolerant cultivars possess a mechan-ism that excludes Al from root apices (Rinco n and

Gonzales, 1992; Samuels et al., 1997).

1. Morphological Changes of Intact Roots andRoot Cells Under Al Stress

Accumulation of coating materials on the epidermis of

the apex and around the cap is commonly observed

upon Al stress. Root meristem cells of canola

(Brassica napus var. napus L. cv. barassa) plants,

grown under control conditions, responded differently

from the much larger control cap cells (Clune and

Copeland, 1999). Under mild Al stress (20M, 24 h),

these cells expanded and increased in number, but

under more severe treatment (80M Al, 24 h) they

diminished in size and number. Furthermore, the dis-

tinct boundary between cells in the root cap meristem

and the elongation zone was no longer apparent, and

the outer layer of cells in the root cap appeared to be

only loosely attached. After only 4 h in 80M Al,

many ultrastructural changes were evident in periph-

822 Matsumoto

Table 1 Extent of Acid Soils in the World and Selected Regions a

Distribution class Global

Region

Central

America

South

America Africa AsiabAustralia/

New Zealand

North

America Europe

Acid land area (106 ha) 3,950 37 917 659 532 239 662 391

Acid land area (%)c

30 35 14 22 76 30 30 37aVon Uexkull and Mutert (1995).bExcluding South and East Asia.cIce-free land area of the globe.

Source: Baligar et al. (1998).


Al (Fig. 1). The root apex (root cap, meristem, and

al., 1997b) (Fig. 2). These results suggest that (1) the


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eral root cells, including the appearance of numerous

small vacuoles that occupied most of the cytoplasm.

After 24 h, disorganization of the cellular contents of

the peripheral root cap cells became obvious, and the

plasma membrane was clearly separated from the cell

wall. The cytoplasm was markedly reduced in volume

and extensively vacuolated. Similarly, Al-induced

vacuolations have been observed in several plant spe-cies (Ikeda and Tadano, 1993; Marienfeld et al., 1995).

In Lemna minor, the vacuolation and numerous mye-

linlike whorls of membrane increased in the apical

meristem of the root (Severi, 1991). Moreover, the vesi-

cles produced by the Golgi apparatus were larger.

However, whether vacuolation is related to the storage

of Al remains to be determined.

Al has a localized effect on auxin transport and

unilateral application of Al inhibited root curvature.

Inhibition of cell elongation in the elongation zone is

the major outcome of the inhibition of root elongation.Shortening of the root elongation zone by Al is accom-

panied by an increase in the diameter and a decrease of

the length of the cells in the second and third layers of

the cortex of the elongation zone of Atlas 66 plants

(Matsumoto, 2000; Sasaki et al., 1996)

The ratio of length to diameter of the cells in the con-

trol root was three to four times larger than that in the

Al-treated roots, and cells in the second and third

layers of the cortex were swollen laterally.

The Al-induced inhibition of longitudinal cell

expansion and cell swelling in the elongation zone

might be related to the disorder of the cytoskeletalnetwork. The orientation of the microtubules (MTs)

is closely related to cell expansion. Longitudinally

elongating cells have transversely oriented MTs. MT-

disrupting agents promote lateral expansion but inhibit

longitudinal expansion. Cortical MTs are known to be

involved in the orientation of cellulose microfibrils.

Indeed, the disappearance of the cortical MTs in elon-

gating cells of wheat roots that was observed under Al

stress (Sasaki et al., 1997a) might be responsible for

these changes in cell growth. Moreover, the time-

dependent effect of Al on MTs stability was correlated

with that on the reduction of root growth.

The effect of Al on the behavior of structural pro-

teins has been investigated intensively in recent years.

The actin network plays an important role in the plant

cell. Al induced a significant increase in the tension

within the transvacuolar actin network in soybean

cells (Grabski and Schindler, 1995). Al resulted in a

reorganization of MTs in the inner cortex, but not

outer cortex, and in the epidermis of the elongation

zone of Zea mays (Blancaflor et al., 1998). They also

Aluminum Stress 823

Figure 1 Time course for wheat root elongation of Atlas 66

and Scout 66. Seedlings were in the presence and absence of 5

(Scout 66) or 50 (Atlas 66) M Al. Data are means (SE) of

results from 10 roots. (From Sasaki, 1996.)

Figure 2 Hematoxylin-stained wheat roots of Atlas 66 and

Scout 66. Seedlings were grown in the presence and absence

of Al for 48 h. (From Sasaki et al., 1997b.)


(Figs. 3, 4).


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824 Matsumoto

Figure 3 Photomicrographs of longitudinal sections of wheat roots of Atlas 66. Roots were treated with or without 20 M Alfor 24 h. Bar indicates 0.2 mm. (From Sasaki et al., 1996.)

Figure 4 Effects of Al on the lengths and diameters of wheat root cells in the second layer from surface in Atlas 66. Roots were

treated with or without 20M Al for 24 h (a) or 48 h (b). Data are means (SE) of results from five or six samples. (From Sasaki

et al., 1996.)



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found that the auxin-induced reorientation and cold-

induced depolymerization of MTs in the outer cortex

were blocked by Al, suggesting that Al increased the

stability of MTs in these cells. The changes of behavior

of MTs against Al stress may depend on the growth

phase of the cells (Sivaguru et al., 1999). This stability

effect of Al in the outer cortex coincided with growth

inhibition. Aluminate [AlOH4 ] at pH 10.0 inducedthe bending of roots of salt-tolerant grass

(Thinopyrum bessarabium A. Lo ve). The roots under

aluminate treatment displayed a number of morpholo-

gical and structural malformations (Eleftheriou et al.,

1993). The root cap decreased in size, and both the

calyptrogen and the meristemic region occupied a

smaller area. Amyloplasts in the root cap cells were

hardly distinguishable and showed less evidence of

sedimentation.

The swollen cells of wheat roots were characterized

by the drastic accumulation of lignin on their cell wallsunder Al stress (Sasaki et al., 1996). The decrease of

cell viability of the elongation zone of wheat roots were

also observed after 3 h of exposure to Al which coin-

cided with the time required for the inhibition of the

root elongation as well as for lignin deposition (Sasaki

et al., 1997b). It is still unknown whether lignin deposi-

tion is involved in the mechanism of Al toxicity. The

morphological changes in roots were characterized by

the cracks on the root surface (Sasaki, 1996) (Fig. 5).

Cracking might be caused by the outward pressure of

the cells in the second and third layers of the cortex of

wheat roots. The distal part of the elongation zone ofmaize roots, where cells are undergoing a preparatory

phase for rapid elongation, is the primary target of Al

influence (Sivaguru and Horst, 1998).

To understand the primary event of Al toxicity, we

must know how the cells in the specific zones, i.e.,

elongation and/or transition zone of the root, accumu-

late Al and how their elongation is inhibited by ultra-

structural alterations. Why do such events cause death

of the cell? In other words, are the matured root cells

resistant to Al toxicity after their elongation has

ended?

2. Inhibition of Cell Division

Cell division in root meristems of several plants is

inhibited by Al (Clarkson, 1965; Morimura et al.,

1978). However, cell division accounts for only 12%

of the overall root elongation. Furthermore, cell cycle

in plants takes about 1 day. However, the primal phe-

nomenon of Al toxicity is the inhibition of root elon-

gation that occurs within hour(s) of Al treatment.

Thus, attention has been largely paid to the inhibitionof root cell elongation as the primary site of Al toxi-

city.

On the other hand, the lethal consequence of Al

toxicity might be inhibition of cell division

(Matsumoto, 2000). Large amounts of Al accumulate

in the developing lateral roots of pea roots where cells

are actively dividing (Matsumoto et al., 1976a). Al was

detected in nuclei of root hair cells by staining and by

chemical determination of Al in purified nuclei pre-

pared from Al-treated pea roots. Furthermore, Al

accumulation in the nuclei of soybean root tips was

detected with Al-sensitive stain lumogallion and con-

focal laser scanning microscopy (Silva et al., 2000). The

nuclei isolated from pea roots treated with 1 mM AlCl3at pH 5.5 for 1 day were fractionated; 73% of the total

Al in nuclei was recovered in the chromatin fraction,

and 94% of Al in chromatin was recovered in DNA

(Matsumoto et al., 1977b).

Expression of genetic information of DNA is regu-

lated by structural changes of DNA and chromatin.

Unwinding of double strands of DNA is a prerequisite

Aluminum Stress 825

Figure 5 Photographs with scanning electron microscope of

wheat root surface of Atlas 66. Roots were treated without

and with 50M Al for 4 h in the presence of 0.1 mM CaCl2.

(From Sasaki, 1996.)



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for expression of genetic information, but separation

of double strands of DNA was interrupted by Al (Fig.

6) (Matsumoto, 1991). Furthermore, the structural

change of chromatin in pea roots treated with Al in

vivo implied that Al induced the condensation and/or

aggregation of chromatin (Matsumoto, 1988). These

results suggested that the template activity of DNA

and/or chromatin of pea roots for RNA synthesis isrepressed by Al. This is caused by Al-induced struc-

tural alteration of DNA and chromatin through the

association of the negative charge of phosphates of

DNA and positively charged Al3 (Morimura and

Matsumoto, 1978). Plant cells require dynamic cytos-

keleton-based networks for various cell activities (e.g.,

differentiation and division). With suspension of

tobacco cells, Sivaguru et al. (1999) found that the

actively dividing log-phase cells were characterized

with faint and larger phragmoplasts and unusually

enlarged daughter nuclei after 6 h of Al treatment.

After a 24-h treatment, no phragmoplasts and spindle

MTs (SMT) from cells having metaphase plate chro-

mosomes were observed.

The disintegration of SMT and disorganization of

phragmoplasts caused by Al might block cell divisiondirectly at the metaphase. As mentioned before, inhibi-

tion of cell division will be the cause for the complete

inhibition of root elongation by Al and subsequent

death. Therefore, elucidation of the detailed mechan-

ism of cell division inhibition by Al will be required in

order to understand the mechanism of Al toxicity

(Matsumoto, 2000).

C. Site of Al Toxicity

1. Apoplast

Although there is disagreement with regard to the site

of Al toxicity, namely, symplastic or apoplastic, many

investigators have stated that 3090% of the absorbed

given to the role of CEC in connection with Al accu-

mulation in the apoplast. On the one hand, high CEC

will be associated with large quantities of Al accumu-

lated in the apoplast. On the other hand, high CEC

may prevent Al from entering the symplast, where it

exerts its lethal effect. However, a clear relationship

between root CEC and Al sensitivity and/or tolerancewas not found across a wide range of plant genotypes

(Grauer, 1992).

As to the binding site of Al in the apoplast, pectin

carboxyl was suggested as a plausible candidate

although almost no evidence has been found to show

the binding of Al to pectin in vivo (Matsumoto et al.,

1977a; Horst, 1995). Although Al is bound by the

negative charge of pectin, the binding capacity of pec-

tin varies with the plant species, and the pectin content

is extremely different between monocots and dicots.

Even in the same species, the pectin content of the

roots differs with the position on the root or with the

chemical modification of pectin, such as methylation

or demethylation changes with the physiological activ-

ity of the cell. A Ca-pectate membrane was used as a

model system. There was a rapid reaction between Al

and Ca pectate, but there was no difference in Al

remaining in solution even after 16 min. Only a slight

decrease was observed after 24 h. The solution contain-

ing 29M Al and 1 mM Ca reduced the flow through

the Ca pectate membrane by > 80% compared to the

826 Matsumoto

Figure 6 Proposed mechanism of inhibition of the tran-

scription of RNA by Al. (A) Normal transcription of RNA

on a sense strand of DNA template in the absence of Al. A

short section of the double strands must open and, thus, only

the sense strand can act as the template. (B) Transcription is

inhibited in the presence of Al. Sections of the double strands

(indicated by two arrows) are captured by Al polymers with

various structures shown as Al Al Al, through the

strong electrostatic interaction between phosphate groups

with a negative charge and the large positive charge of the

Al polymer. Thus, separation of the strands is blocked and

limited synthesis of RNA results. In addition, the Al polymer

with its large positive charge on one helix, shown as nAlm

where n and m are highly variable and depend on coexisting

factors, can associate with the other helix and cause aggrega-

tion of chromatin fibers. (From Matsumoto, 1991.)


Al is localized in the apoplast (cf. Tice et al., 1992;

Rengel, 1996). Two possible interpretations has been


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solution containing 1 mM Ca only. These results sug-

gest that an important effect of toxic Al is a reduction

in water movement into roots (Blamey et al., 1993).

Interactions of Al with other cell-wall components,

such as enzymes, extensin and xyloglucan may affect

the functional integrity of cell walls. In the roots of

cotton seedlings, Al impaired the sucrose utilization

for cell wall formation (Huck, 1972). Al also inducedthe production of cell-wall components of squash seed-

lings, especially of hemicellulose, (Van et al., 1994).

Al stress increased the level of covalently bound cell

wall proteins in pea roots (Pisum sativum cv. Alaska).

In vitro and in vivo Al binding experiments have

suggested that extensin has the highest capacity to

bind Al among cell wall proteins (Kenjebaeva et al.,

2001).

2. Plasma Membrane

The plasma membrane is one of the first targets of Al

(Haug, 1984; Matsumoto, 2000). Al binds readily to

the plasma membrane because of its high content of

phosphate such as phospholipids and the negative

charge of the membrane surface. Al3 has a 560-fold

higher affinity for the phosphatidyl choline surface

than Ca2 (Akeson et al., 1989). Structural and func-

tional changes of the plasma membranes are induced

by Al binding, although the evidence of Al binding to

the plasma membrane in vivo is limited (Matsumoto et

al., 1992). Al-induced changes in membrane behavior

of intact root cortex cells ofQuercus rubra root showedthat Al altered the activation energy required to trans-

port water (32%), urea (9%), and monoethyl urea

(7%) across cell membranes as measured by the plas-

mometric method (Zhao et al., 1987). Al increased the

lipid partiality of the plasma membrane at > 9C but

decreased it at temperatures < 7C. These changes in

membrane behavior are explainable if Al reduces mem-

brane lipid fluidity and kink frequency and increases

packing density and the occurrence of straight lipid

chains (Chen et al., 1991). It can be concluded that

Al3 (1) increased membrane permeability to the none-

lectrolytes, (2) decreased the membrane partiality for

lipid permeators, and (3) decreased membrane perme-

ability to water caused by increased activation energy.

It is thus implied that Al3 alters the architecture of

membrane lipids (Vierstra and Haug, 1978).

Furthermore, Al3 inhibited the influx of cations

and enhanced the influx of anions. This was caused

by the Al-induced formation of positively charged

layer at membrane surface influencing ion movement

to the binding sites of the transport proteins. A posi-

tively charged layer would retard the movement of

cations to the plasma membrane. This is explained

by the charge of the membrane surface potential,

Zeta potential, through the binding of Al. It was also

argued from the relationship between Al tolerance and

surface negativity of plasma membranes (Wagatsuma

et al., 1995). Al reduced the negative charge associated

with phospholipids, i.e., depolarization of Zeta poten-tial, and proteins by binding to these charged groups

or shielding the surface charges. Alteration of K

efflux and H influx by Al also affect the Zeta potential

as well as the potential difference (PD) across plasma

membrane (Sasaki et al., 1994a).

A more direct effect of Al3 is its binding to trans-

port proteins and impairs their function. For instance,

Al3 blocks inward-rectifying K channels in root

hairs of wheat (Triticum aestivum) and VDAC, chan-

nel-forming protein located in the outer mitochondrial

membranes (Dill et al., 1987). Recently, a special inter-action was found between depolarization of Zeta

potential and decrease of H

-ATPase of plasma mem-

brane of squash roots treated with Al. The interaction

was typically observed at root tips, 05 mm portion of

the root (Ahn et al., 2001). However, the varietal sen-

sitivity to Al3 is not based on the difference in cell

surface electrical potential (Kinraide et al., 1992), and

inhibition of root growth by Al is not caused by the

reduction in current or H+ influx at the root apex

(Ryan et al., 1992). On the other hand, a different

membrane potential depolarization of root cap cells

preceded Al tolerance in snapbean (Phaseolus vulgarisL.) (Olivetti et al., 1995). The Al-tolerant cultivar Dade

depolarized rapidly upon exposure to Al, but the Al-

sensitive cultivar Romano was only slightly depolar-

ized. This might be related to the fact that Al reduces

the K efflux channel conductance in the tolerant

Dade root cap cells, but does not affect it in the sensi-

tive cultivar Romano. Further research is needed to

understand the interrelationship between Al toxicity

and/or tolerance and electrophysiology.

One of the biochemical changes of the plasma mem-

brane is the Al-dependent lipid peroxidation in the

root tip of soybean (Glycine max). A close relationship

existed between lipid peroxidation and inhibition of

root elongation induced by Al and/or Fe toxicity

and/or Ca deficiency (Cakmak and Horst, 1991).

Enhanced lipid peroxidation by oxygen free radicals

can be a consequence of primary effects of Al on mem-

brane structure. The tolerance mechanism against Al

toxicity in terms of lipid peroxidation was proposed for

tobacco suspension cells (Yamamoto et al., 1998).

Lipid composition can be a determinant of the varietal

Aluminum Stress 827



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in the free Ca2 concentration and triggering callose

synthesis cannot be excluded.

Ca2 may not be the only signal for callose forma-

tion, and alteration in the plasma membrane architec-

ture might also be important for callose synthesis

(Jacob and Northcote, 1985). As callose is released

into the apoplast after its synthesis on the plasma

membrane, cell walls of root cells of Al-treated plantsmost likely contain callose depositions.

What is the inhibitory function of callose under Al

stress? Callose can be considered as a sealing system

in plants. Callose is localized in the wall around the

plasmodesmata, which appear to be structurally sub-

divided. Thus, the constricting force upon callose

synthesis would be transmitted to the plasmodesmal

core (Turner et al., 1994). This will inhibit the trans-

port of cellular compounds through plasmodesmata

under Al stress. A large increase of callose accumula-

tion at the plasma membrane under Al stress wasfound in Al-sensitive wheat roots. Such an increase

inhibited the cell-to-cell trafficking of molecules

through the plasmodesmata, resulting in the inhibi-

tion of root elongation. Furthermore, these events

are markedly repressed in the presence of 2-deoxy-

D-glucose, which is a callose synthase inhibitor

(Sivaguru et al., 2000). However, in an Al-tolerant

Arabidopsis mutant, no direct relationship between

Al uptake and callose formation was established

(Larsen et al., 1996).

D. Signal Transduction and Al Signal

Plants respond to Al stress quickly. Inhibition of root

elongation is observed within less than an hour, and

homeostasis of cytoplasmic free Ca2 is broken

instantly after Al addition. Special attention has been

paid to phosphoinositide-associated signal transduc-

tion. AlCl3 and Al-citrate inhibited phospholipase C

(PLC) of the microsomal membrane in a dose-depen-

dent manner in wheat roots. I50 was observed at

1520M Al (Jones and Kochian, 1995). Binding of

Al to microsomes and liposomes was found to be lipid

dependent, with the signal transduction element PIP2having the highest affinity for Al with an Al:lipid stoi-

chiometry of 1:1. These results suggest an Al effect on

the signal transduction pathway that is associated with

the mechanism of Al toxicity.

How is the Al signal recognized by receptor and

how is it transported into the cytoplasm at the root

apices? Bennet and Breen (1991) proposed that the

Al signal is perceived in the root cap of Zea mays.

Matsumoto et al. (1996) speculated that the transduc-

tion of Al signal in barley roots is related to an increase

of ABA. ABA induces both the ATP- and PPi-depen-

dent H pump activity of the tonoplast (Matsumoto et

al., 1996). Contrary to ABA, transport of exogenously

applied [3H]indole-3-acetic acid to the meristemic zone

was significantly inhibited by Al in maize roots. The

signaling pathway in the root apex mediating the Al

signal may be responsible for the genotypic differencein Al resistance (Kollmeier et al., 2000). The biochem-

ical mechanism in terms of the transduction of Al sig-

nal is poorly understood. Protein phosphorylation may

be involved because of the strong association of Al

with phosphate.

E. General Metabolism Affected by Al

The effect of Al on excised root apices and isolated

mitochondria of wheat was investigated. O2 uptakeby excised roots was reduced by 23% and 35%

after 12- and 24-h treatment with 75M Al.

Mitochondria isolated from Al-treated roots had

reduced oxidative capacity with supply of electrons

to complexes I and II. It was found that initially Al

affected electron flow through complexes I and II,

and after longer exposure interacted with other sites

in the mitochondria (de Lima and Copeland, 1994).

Al tolerance of Phaseolus vulgaris (cv. Dade) was an

inducible trait. In this cultivar, the resumption of root

elongation during recovery from Al treatment was

accompanied by increased rates of respiration.Respiration rates slowly declined over the 72-h treat-

ment of Al-sensitive Romano. When partitioned into

growth and maintenance expenditures, a larger pro-

portion of root respiration of Al-treated Dade plants

was allocated to maintenance processes, potentially

reflecting diversion of energy to metabolic pathways

that offset the adverse effects of Al toxicity (Cumming

et al., 1992).

Carbon metabolism is also affected by Al. Al stress

increased alcohol dehydrogenase activity in wheat

(Triticum aestivum cv. Vulcan) roots. Sucrose synthase

and lactate dehydrogenase were also increased in Al-

treated roots, suggesting that the early effect of Al on

wheat roots may be a shift from aerobic to anaerobic

metabolism. The first two enzymes in the pentose phos-

phate pathway (G-6-PDH and 6-PGDH) decreased in

Al-sensitive wheat cv. Grana. However, these two

enzymes first increased, but then decreased in Al-toler-

ant rye (Sla ski et al., 1996). These results suggest that

the mechanism of Al resistance involves the regulation

of the pentose pathway.

Aluminum Stress 829



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III. ALUMINUM TOLERANCE MECHANISM

A. Al Tolerance on Genetic Basis

The bimodal distribution of phenotypes corresponding

to 3:1 segregation ratio for Al tolerance/sensitivity in

populations derived from crosses between tolerant and

sensitive cultivars revealed the presence of single majorgenes for Al tolerance (Gain, 1998). In addition to

major genes conferring major differences in Al toler-

ance, there is also some evidence that minor genes or

modifier genes may play a role in modulating the effect

of major Al tolerance genes. Other studies have sug-

gested that genetic factors located on the long arm of

chromosome 2D prevent accumulation of Al in root

apical meristems of the BH1146 euploid wheat (Aniol,

1995). However, other genetic factors are also located

on these chromosome segments that control Al detox-

ification in the root tips of Al-tolerant lines.

The D genome of wheat may determine the toler-ance to acid soil and consequently contribute to the

increased adaptation of hexaploid wheats during

their evolution. Atlas 66 is a well-known Al-tolerant

cultivar of wheat. However, not all the genes for tol-

erance to Al in Atlas 66 are located on the D genome

chromosome (Berzonsky, 1992). Furthermore, Al tol-

erance in wheat is a dominant trait and the majority of

observed variability could be explained by hypotheses

of two or three gene pairs, each gene affecting the same

character with complete dominance of each gene pair.

Al tolerance in the ditelosomic line of Chinese Spring

wheat cultivar revealed that genes controlling this

character are located on the short arm of chromosome

5A and the long arm of chromosomes 2D and 4D

(Delhaize et al., 1993a).

Conservation of the Al tolerance gene by various

species was investigated. RFLP markers for a major

wheat Al tolerance gene AltBH were found on the long

arm of chromosome 4D, while in rye, the Al tolerance

gene was located on chromosome 4, which harbors

chromosome segments homologous to regions of

wheat chromosome 4D. In barley, the Al tolerance

gene Alp is almost certainly orthologous to the wheatAltBH gene due to the fact that the relative positions of

Alp and AltHB with respect to a common set of mole-

cular markers are virtually identical in both genomes

(Berzonsky, 1992). In spite of efforts made so far, the

genetics of Al tolerance is little known for any single

species. As to the physiological functions of Al toler-

ance genes, Delhaize et al. (1993b) found that Al tol-

erance, controlled by the Alt gene in wheat, appeared

to be dominant across a range of Al concentrations

based on identical Al tolerance in heterozygotes and

Alt 1 homozygotes. This gene controls the excretion of

malate upon Al stress.

B. Genetic Basis of Al Tolerance

Application of lime to acid soils to increase the soil pHis one strategy for alleviating Al toxicity. However, this

technique is problematic from the economical and

environmental points of view. Another strategy is to

use Al-tolerant crops. Breeding may depend on classi-

cal techniques and/or transgenic plants to which Al

tolerance genes are being introduced. Snowden et al.

(1995) isolated several cDNA (wali 17) whose tran-

script accumulates in wheat under Al treatment. A

cDNA library constructed from the mRNA of Al-trea-

ted roots of Al-sensitive wheat (cv. Victory). It was

screened with a degenerate oligonucleotide probe

derived from a partial amino acid sequence of theAl-induced protein TAl-18. Out of seven clones that

initially hybridized with the probe, one encoding a

novel 1,3--glucanase mRNA was upregulated in Al-

treated roots, with highest expression after 12 h. A

second cDNA showed similarity to genes encoding

cytoskeletal fimbinlike protein. Unfortunately, a trans-

genic protein enriched with those genes was not con-

structed (Cruz-Ortega et al., 1997). Al ions bind to

phospholipids, and the plasma membrane is a primary

barrier to the entry of Al into the cells (Matsumoto,

1988; Kochian, 1995). Thus, a change in lipid compo-

sition of the plasma membrane could improve the

resistance of the cell by excluding Al (Delhaize et al.,

1999). Cloned wheat cDNA (TaPSS1) that codes for

phosphatidyl serine synthase (PSS) was tested.

Overexpression of PSS increased Al resistance in

yeast. However, a high level of TaPSSI expression in

Arabidopsis and tobacco led to the appearance of

necrotic lesions on leaves, which may have resulted

from the excessive accumulation of PS (Delhaize et

al., 1999). An Arabidopsis blue-copper-binding protein

gene, a tobacco glutathione S-transferase gene, a

tobacco peroxidase gene, and a tobacco GDP dissocia-tion inhibitor gene conferred a certain degree of resis-

tance to Al (Ezaki et al., 2000). These lines also showed

increased resistance to oxidative stress, suggesting a

link between Al stress and oxidative stress in plants.

One successful approach was the generation of

transgenic tobacco and papaya with the citrate

synthase (CS) gene from Pseudomonas aeruginosa

with the 35S promoter of cauliflower mosaic virus

introduced using a Ti plasmid derived from a transfor-

830 Matsumoto



11/18

mation system. The idea was that organic acids serve

as chelating agents and may prevent Al toxicity

(Delhaize and Ryan, 1995; Fuente et al., 1997).

Hematoxylin staining was employed to examine

whether the increased Al tolerance of the CSb lines is

due to the inhibition of Al uptake by the root tip.

Following exposure to Al CSb lines showed a consid-

erably lighter staining than the control. Apparently,the expression of a citrate synthase in the cytoplasm

increases the concentration of citrate, which led to a

higher rate of its efflux; the higher synthesis and excre-

tion of citrate confers Al tolerance.

C. Organic Acids as Al-ChelatingSubstance

Since higher plants cannot move away from the acid

soil, they have developed ways to reduce this edaphicstress. An effective strategy to reduce the stress is to

chelate the Al3

in the rhizosphere and by that reduce

its toxicity. Exclusion of malate from wheat (Delhaize

et al., 1993b); citrate from maize, Cassia tora, and rye

(Miyasaka et al., 1991; Zheng et al., 1998; Li et al.,

2000b); and oxalic acid from taro and buckwheat

(Ma and Miyasaka, 1998; Ma et al., 1997b) have

been reported. Some plants exclude both malate and

citrate under Al stress. Malate and oxalate are excreted

instantly under Al stress, but citrate is excreted only

after a lag phase. These results suggest that stored

malate and oxalate are excreted while citrate is synthe-sized by a gene-regulated system.

The organic acids were classified into three groups

of Al detoxifiers: (1) strong (citrate, oxalic, tartaric);

(2) moderate (malic, malonic, salicylic); and (3) weak

(succinic, lactic, formic, acetic phthalic) (Hue et al.,

1986). The following facts support the role of excreted

organic acids as detoxifiers; During the first 20 h of Al

exposure, the root growth rate of both tolerant and

sensitive maize varieties was severely inhibited.

However, after this period, root growth was resumed

in the tolerant plants, but remained severely inhibited

in the Al-sensitive one. A dose-dependent citrate and

malate exudation was observed from tolerant but not

from sensitive roots (Jorge and Arruda, 1997; Yang et

al., 2000). The root of the Al-resistant snapbean

released 70 times more citrate in the presence of Al

as in its absence, and the amount of citrate excreted

was 10 times as much as that of Al-sensitive cultivars

(Miyasaka et al., 1991). Similar results regarding

malate exclusion were obtained with Al-tolerant and

Al-sensitive isogenic wheat lines.

In > 36 lines of wheat cultivars differing in Al resis-

tance that were screened, Al-stimulated malate release

was correlated with Al resistance (Ryan et al., 1995). It

was shown that addition of organic acids to the solu-

tion ameliorated Al toxicity in the root of Al-sensitive

varieties and reduced dramatically the loss of viability

of root cells (Li, 2000). This may be explained by the

fact that complexes of Al with di- and tricarboxylicacids were not transported through root cell mem-

branes (Ma et al., 1998). Excretion of organic acids is

Al3 specific and is not induced by other trivalent

cations (Ma et al., 1997b).

The loss of a certain chromosome arm resulted in a

decrease in Al resistance in ditelosomic wheat lines and

decreased rates of root apical malate release concomi-

tant with decreased Al exclusion (Kochian, 1998).

Exposure to Al induced depolarization of the mem-

brane potential (Em) in Al-tolerant wheat cultivars

but not in an Al-sensitive cultivars. Depolarizationwas specific to Al. Al-induced depolarization of root

cap cell membrane potentials is probably linked to

malate release (Papernick and Kochian, 1997).

Al3 triggers the opening of the putative malate-

permeable channel. Several antagonists of anion chan-

nels inhibited the Al-stimulated efflux of malate. The

anion channel antagonist niflumate inhibited the cur-

rent in whole-cell measurements by 83% at 100 M Al.

Patch clamp recordings revealed a multistate channel

with single-channel conductance of between 27 and

66 ps. This is a good candidate to be the transport

system facilitating Al-induced malate release (Ryan etal., 1997b). K-252a, a potent inhibitor of protein phos-

phorylation, reduced dramatically the excretion of

malate from Al-treated wheat, suggesting the involve-

ment of protein phosphorylation for the regulation of

malate excretion under Al stress (Osawa and

Matsumoto, 2001).

Transgenic introduction of the bacterial cytosolic

citrate synthetase gene into tobacco and papaya

resulted in Al tolerance (Fuente et al., 1997).

Excretion of organic acids from the root apex where

Al injury is located seems to be a reasonable strategy

for the effective use of carbon and energy by the plant

(Delhaize and Ryan, 1995). Such experimental evi-

dence indicates that the mechanism of Al exclusion

that depends on the chelation of Al3 with excreted

organic acids is an effective strategy. However, it is

unclear whether the quantities of organic acids released

are adequate to explain the insensitivity to Al of the

more tolerant genotypes. Consumption of the excreted

organic acids by soil bacteria should also be consid-

ered. The question why different plant species excrete

Aluminum Stress 831



12/18

different organic acids in different ways upon Al stress

also remains to be solved. The efflux of organic acids is

probably switched on or off by the Al3 stress, because

continuous excretion of organic acids from roots

would consume carbon and energy in extraordinary

amounts. The precise mechanism remains to be eluci-

dated, and a more integrative, multifaceted model of

tolerance is needed (Parker and Pedler, 1998).The role of intracellular organic acids on Al toler-

ance was investigated using the Al-tolerant plant

Hydrangea macrophylla. Leaves of hydrangea may

contain up to 15.66 mmol Al kg1 fresh weight.

About 77% of the total Al exists in the cell sap (Ma

et al., 1997a). The ligand in the Al complex of hydran-

gea leaves was citric acid with a molecular ratio of 1 Al

to 1 citrate. The purified Al:citrate complex from

hydrangea leaves did not inhibit the root elongation

of maize and did not decrease the cell viability

although both parameters were strongly inhibited bythe same concentration of AlCl3. This means that Al in

the form of Al:citrate is not toxic. Also, buckwheat

contains large amounts of Al compared to other crop

plants but can grow normally under Al stress. This

suggests that buckwheat can detoxify Al. The measure-

ment of 27Al-NMR revealed that the Al in buckwheat

leaves and roots existed as an Al:oxalate (1:3) complex.

The Al:oxalate (1:3) complex is the least toxic complex

compared to other complexes. Buckwheat contains a

large amount of oxalate regardless of Al stress and can

excrete oxalate immediately after the plant is exposed

to Al. Even if some Al is incorporated into the planttissues, it is immediately chelated there.

D. Protein Expression in Roots Under AlStress

Wheat genotypes differing in Al tolerance were com-

pared for qualitative and quantitative differences of

their proteins. However, no conclusive evidence for

upregulation of a certain protein that confer Al toler-

ance was reported (Delhaize et al., 1991; Ownby and

Hruschka, 1991). Most of the changes in protein

expression associated with Al stress probably result

from the effects of Al on cell metabolism. Another

approach was to look for proteins that are character-

ized by their binding capacity to Al. Appearance of the

23-kDa peptide in root exudates cosegregated with the

Al-resistant phenotype in F2 populations and had a

significant Al-binding capacity (Basu et al., 1999).

Similarly, a 51-kDa membrane-bound protein accumu-

lated in the root tip of Al-tolerant wheat (PT741)

under Al stress. The specific induction of the 51-kDa

band in PT741 suggested a potential role of these pro-

teins in mediating resistance to Al, associated with the

tonoplast. Antibodies raised against tonoplast H+-

ATPase and H+-PPiase did not crossreact with 51-

kDa protein, although Al stress induced these enzymes

in the barley roots (Matsumoto et al., 1996; Taylor et

al., 1997).

E. Mucilage

The root apices of most plant species are covered by

mucilaginous substances that are excreted from root

volume). The meristem and cap region where Al

toxicity is dominant are coated with mucilage that

ranges in thickness from 50m to 1 mm. Mucilage

consists mainly of polysaccharides > 2 106 daltons.

Abundant sugars are glucose, galactose, and arabi-

nose. Uronic acids are smaller in amount but charac-teristic of the mucilage. Mucilage has various

protective functions against toxic metals in the soil

and has a high Al-binding capacity. Fifty percent of

the total Al of root apices of cowpea was associated

with mucilage (Horst et al., 1982). Al bound to muci-

lage of wheat roots accounted for $ 2535% of the Al

remaining after desorption by citric acid. The Al in

rhizosphere is bound to mucilage that blocks the

entry of Al into the root. When the mucilage was per-

iodically removed from the root tips of cowpea with a

brush, inhibition of root elongation was increased.

Apparently, binding of Al to mucilage is a mechanismof Al tolerance. A good correlation exists between

mucilage volume and Al tolerance. Organic acids

released into a mucilage droplet would diffuse slowly;

thus, the mucilage droplet would form a region of high

concentration of organic acids where Al is captured

before reaching the root surface.

However, a protective role of the mucilage against

Al injury is difficult to reconcile with the lack of evi-

dence that mucilage excretion affected by Al. On the

contrary, disappearance of mucilage is one of the first

visible symptoms of Al toxicity (Puthota et al., 1991).

The role of mucilage in the protection of roots against

Al toxicity depends on the amount of mucilage

excreted and how strongly Al bounds to mucilage.

Mucilage from maize roots is strongly bound to Al

but failed to prevent Al-induced inhibition of root

elongation (Li et al., 2000a). Approximately 50% of

the total Al of the root apices was located in the muci-

lage of cowpea, while only 922% of Al in maize root

was bound to mucilage. The binding is decreased by

the lower content of uronic acids (3%) in maize muci-

832 Matsumoto


cap cells (see also Chapter 3 by Sievers et al. in this


13/18

lage as compared to 11.5% in cow pea mucilage (Li et

al., 2000a). It will be necessary to determine the Al-

binding sugar component in mucilage as well as total

amount of mucilage and kinetic data of synthesis and

excretion of mucilage in order to understand the role

of mucilage in Al resistance.

F. pH in the Rhizosphere

Solubility of Al depends strongly on pH. Thus, main-

tenance of a high solution pH may reduce the solubi-

lity and toxicity of Al. An increase in the pH of dilute

nutrient solution from 4.5 to 4.6 caused a 26% decline

in soluble Al concentration (Blamey et al., 1983). This

suggests that even a slight pH change can affect the

toxicity of Al as well as tolerance. However, measure-

ment of pH should be done carefully because the

change of pH near the root surface is important. For

this purpose, a vibrating microelectrode was used tomeasure pH at a radial distance of 20 and 50m

from the surface of the root tip of a wild-type and of

an Al-tolerant Arabidopsis (Degenhardt et al., 1998).

The Al-tolerant Arabidopsis mutant alr-104 showed a

clear increase of pH at the rhizosphere in the presence

of Al, but the wild type did not. Al exposure of alr-104

induced a twofold increase in net H influx localized at

the root tip. The increased flux raised the root surface

pH of alr-104 by 0.15 unit, suggesting that Al resis-

tance in alr-104 is mediated by pH change in the rhizo-

sphere. Difference in Al resistance between wild type

and alr-104 disappeared when roots were grown in pH-buffered medium. It is interesting that no difference in

root H fluxes between wild type and alr-104 was

detected in the absence of Al.

IV. BENEFICIAL EFFECT OF ALUMINUMON PLANT GROWTH

Plants that contain > 1000 ppm Al are called Al accu-

mulators. Among the 259 plant families, 37 Al accu-

mulator species were found and most of them are

arborescent cryptogams (Chenery and Sporne, 1976).

Al accumulators can grow in acid soils, and growth of

some of them is even promoted. Tea (Camellia sinensis)

may contain as much as 30,000 ppm Al in old leaves

but only 600 ppm in young leaves. The specific loca-

tions of Al in epidermis of old leaves and in the cell

lumen of bean and barley root was demonstrated by

the staining of Al with aluminon and by x-ray (EMX)

microanalysis (Waisel et al., 1970; Matsumoto et al.,

1976b). The secondary cell wall of epidermal cells of

old leaves is thicker and Al may have accumulated in

the cell wall during thickening. The formation of new

roots was greatly accelerated after 1 month of Al treat-

ment and thereafter the growth of tops was positively

affected (Matsumoto et al., 1976b). Tea is a sensitive

plant for phosphate nutrition and markedly inhibited

by the excess of phosphate. Konishi et al. (1985)

showed that maximum growth of tea occurred withcoexistence of Al with phosphate. The reduced growth

of tea at 0.8 mM phosphate was dramatically stimu-

lated by the presence of 1.6 mM Al with new root

formation. Al plays a regulatory role in the effective

absorption and utilization of phosphate. Another

strategy of tea plants against Al toxicity is binding of

most of Al to catechin, phenolics, and organic acids

(Nagata et al., 1992). The mechanism of Al tolerance is

generally carried out by internal detoxification or

excretion of chelators, but the mechanism of beneficial

effect of Al on the growth has not been clearly demon-strated.

V. CONCLUDING REMARKS

Inhibition of root elongation caused by Al toxicity is

one of the most deleterious factors for plant growth in

acid soils. Al3 concentrations as low as 1 mol at pH

4.55.0 inhibit root elongation within 1 h. Absorbed Al

is localized at the root apex, where it inhibits cell func-

tions. It is therefore important to know the effect of Al

on the processes of cell elongation and cell division atthe root apex.

There are several unsolved problems underlying the

mechanism of Al toxicity. The receptor of the Al signal

on root cell membrane and how the signal is trans-

mitted remain unknown. Does the signal work only

in the apoplast? If so, there must be a signal transduc-

tion system through the plasma membrane into the

symplast. Structural proteins like tubulin and actin

are candidates for participation in that system.

Another possibility is that Al itself is active in the sym-

plast. In this case, we must know the mechanism of Al

transport through the plasma membrane.

The role of organic acids, both intracellular and

extracellular, in the mechanism of Al tolerance has

been clarified markedly during the last decade. Al-tol-

erant plants accumulate less Al than sensitive ones, and

formation of chelaters reduces Al toxicity. The major

organic acids are citric, malic, and oxalic acids. Why

do different plant species excrete different organic acids

by the same Al signal? Is there any other Al-chelating

compounds of plant origin other than organic acids? Is

Aluminum Stress 833



14/18

the amount of excreted organic acids sufficient for Al

chelation in the rhizosphere in acid soil? (See also

Much progress has been made in the molecular

aspects of Al binding to oxalic acid and excretion

mechanism of malic acid. However, an important pro-

blem still to be solved is the regulatory mechanism of

the synthesis and excretion of organic acids upon theAl signal. The knowledge of the cell responses to the

short-term effects of Al is expected to help us to under-

stand the whole-plant responses and would lead to the

improvement of crop production under long-term

effects of Al.

ACKNOWLEDGMENT

The author wishes to thank Mrs. S. Rikiishi for her

careful preparation of the manuscript.

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838 Matsumoto

chapter 46 - plant roots under aluminum stress - toxicity and tolerance

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