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Uptake of Aluminium by the root tips of an Al-sensitive and Al-tolerant cultivar of Zea mays
Plant Physiol.
Biochern.,
1998, 36 (h), 463-467
Uptake of Aluminium by the root t&s of an Al-sensitive tolerant cultivar of Zea mays Jos6 Intro’*,
and Al-
Jean BarIoy2, Paul FaBavier3
’ Department of Agronomy, State University of Mating& Av Colombo 5790, Maring&PR, 87020-900, Brazil ’ Laboratoire d’agronomie, Ecole nationale sup&ieure agronomique de Rennes, 35042 Rennes, France. ’ Centre de coopCration internationale en recherche agronomique pour le dirveloppement-URA, BP 5035, 34032 Montpellier, France. * Author to whom correspondence should be addressed 1fax 5.5 44 263 42 42) (Received May 2, 1997; accepted November 13, 1997)
Abstract - Uptake and compartmentation of Al in the roots tips were investigated in two corn (Zecr n~,vs L.) cultivars (HS7777 single sensitive hybrid and CS25-M single tolerant hybrid). Seedlings were grown for 24 h in nutrient solution at pH 4.4 characterized by low ionic strength (2,06 FM) and low Al”+ activity (10.3 vM). The root elongation decreased strongly for the sensitive cultivar. There were no differences in the contents of total Al in the entire roots between cultivars. Nonexchangeable-Al and exchangeable-Al contents were higher in the root rips 01‘ HS7777. The sensitive cultivar also showed higher total and exchangeable-Al contents in excised root tips in 180 min of Al uptake. Al uptake for both cultivars increased in solution containing 0. I mM of 2,4-dinitrophenol. Al contents were higher for the tolerant cultivar. 0 Elsevier, Paris Aluminium I Al distribution / Al uptake I dinitrophenol / root tips I Zea mays Al, aluminium /2,4 DNP, 2,4-dinitrophenol / CEC, cation exchangecapacity / ICP, inductively coupled plasma
1. INTRODUCTION Aluminium (Al) toxicity is one of the principal fattors that limits plant growth and production in acid soils [S]. The first visual symptom of Al toxicity is a reduced root growth [ 12, 14, 201 and the toxic effects are more evident in the root tips [ I, 21. The tolerance of plants against Al toxicity is developed using two basic strategies [ 13, 191. The first strategy, blocking the entry of Al into the symplastic compartment could act through a pH barrier at the rhizosphere, a selective permeability of the plasma membrane, the exudation of chelates, or the immobilization of Al in the cell wall. The second strategy, in the symplastic compartment, acts through Al chelation in the cytosol, vacuolar compartmentation, or formation of complexes with Al-binding proteins. The cell wall and the cell membrane (under normal metabolic conditions) probably function as an important barrier to the passive movement of Al into symplastic compartment 1211. Zhang and Taylor 1251 showed under non-metabolic conditions (e.g. 2,4 diniPlant
Physiol. Biochem., 0981-9428/98/06/O Elsevier, Paris
trophenol in uptake solutions) that the Al concentration in wheat (Tuitic~nz aestiwnz L.) root tips was strongly increased . Al may be adsorbed in the cell wall by the negative charges [22]. Even though the root cation exchange capacity (CEC) plays a role in the process of Al tolerance, the results in the literature are contradictory. Lower CEC values were found in the Al tolerant species [ 191. Blarney et al. [4] investigated the Al tolerance in relation to root CEC of 12 plant species (monocots and dicots) and found that tolerance of Al was not closely associated with differences in root CEC values. The selective permeability to Al and negative charges on the surface of the plasma membrane are important characters which determine the passage of Al into the symplasm [23, 241. The negative charges on the surface of the plasma membrane of root cells are stronger in the sensitive plant species. A larger amount of Al is bound to the plasma membrane, and the plasma membrane is eventually destroyed by Al 1231.
464
J. Pintro
et al.
The nonexchangeable-Al can be defined as the Al in the symplastic compartment and the Al precipitated or chelated in the apoplastic compartment, which: cannot be exchanged. The Al in the apoplastic compartment adsorbed on the negative charges of cell wall is defined as exchangeable-Al [20]. The Al toxicity effects on root growth are less correlated with the total Al content in roots [ 131. The analysis of root tips for total Al, although preferable to analysis of whole roots, does not identify differences in Al distribution within root t@~, which may be important in understanding the basis ot’ Al tolerance [6]. The compartmentation of Al in the root tips [ZO, 251 and the comparison of Al uptake by roots under normal and non-metabolic conditions [21) art: bettel parameters. The goal of this study was to investigate Al uptake and AI compartmentation in the root tip.? of an Al-sensitive and Al-tolerant cultivar of corn.
2. RESULTS
AND DISCUSSION
2.1. Root growth The Al treatment (I 0.3 yM Al’+ activity) reduced the elongation of main root for both cultivars, but root growth of C525-M was less inhibited by Al than HS7777 was @gure I). Root growth was reduced to
withoat
Figure 1. Net elongation and C52S-M (Al-tolerant) without or with 10.3 pM n=3).
Al
of the main root of HS7777 (Al-!,ensitive) corn cultivars cultivated in nutriem solution A?+ activity at pH 4.4 for 24 h (mean f S.E..
69 o/ and 43 o/ofor HS7777 and C525-M, respectively, in relation to control. Tan and Keltjens [18] for sorghum (Sorghum bicolor L. Moench), Guevara et al. [9] and Pintro et al. [ 151 for corn (&a mays L.), and Tice et al. [20] for wheat (Tuiri~m aesrivum L.) have found similar differences between Al-sensitive and Al-tolerant cultivars. 2.2. Aluminium
in entire roots and in root tips
There were no differences in the contents of total Al in the entire roots [S69 + 26 mg-kg-’ for HS7777 and 555 f 12 mg.kg-’ for C525-M (mean + S.E., n=3)] between cultivars. However, the Al contents in the root +.s of C525-M were lower than for HS7777 (figure 2). The AI contents for CS2S-M correspond to 55 % of total Al, 2 I % of exchangeable-Al and 73 ‘II, of the nonexchangeable-Al contents found for HS7777. These results (total Al) are in concordance with previous studies 16, 20, 251 working with an At-tolerant and Al-sensitive cultivar of wheat. Total Al content in the entire roots was not a good indicator of the differences in the Al tolerance of the two cultivars. The results showed that the amount of Al in the root tips was an efficient tool for discriminating between the two cultivars. Analyses of Al content in root tips are in agreement with the hypothesis that CS2S-M encodes a tolerance mechanism by exclusion of Al from root tips 161. 2.3. Aluminum
in excised root tips
Total Al contents in excised corn root tips increased with time lfigure 3 A). Uptake of Al was initially higher in the first period (approximately 30 min) for both cultivars. At the end of Al uptake time (300 min), there was a tendency towards an equilibrium value. Total Al concentrations were higher in the HS7777 than in CS2S-M. Similar results in Al-uptake kinetic were found by Huett and Menary [lo] with cabbage (Rmssictr oleracea L.), lettuce (Lnctuca sutiva L.), kikuyu grass (Penrzisetum clundestinium Chiov.) and by Zhang and Taylor [2S ] with wheat, despite differences in experimental conditions (such as species, pH and Al concentrations). Al-uptake kinetic could be described by an exponential function. The exchangeable-Al contents found for the tolerant cultivar were lower than those found for the sensitive cultivar (figtire 3 B). The differences in the exchangeable-Al between cultivars may reflect, in part, the lower root CEC values of root rips for C.525 M (74 f 4 mmol.kg-’ dry matter) than for HS7777 (92 + 3 mmol.kg-’ dry matter) (mean rt S.E., n=3).
Upbake of Al by the root tips of different
Total
-
Al
900 800 700 600 500 400 300 200 100 0
0
l-
of Zeu muys
465
750 700 650 600 550 500 450 400
1100 1000
cultivars
30
60
90
120
150
180 210
240
270
300
330
Exchangeable-Al 250
150 100 HS7117 = 26.2+225.1 50
Nonexchangerbte-Al’
C525-M 10
800
30 .,
.
-
600
-
500
-
70 .,
90
(1 -e(“.m-))
R’ = 0.99
(I-e(-‘mmpo’)
RZ=0.98
110
.,
a,
130 .,
HS7777 with DNP = 86.7+2.8xtime C525-M with DNP 108.3+3.2xtime =
C 700
50 ,
= -17.5+19X3
With
DNP
/? ,,
,zI
400-
..*(-
,,I’
150 .,
170 a,
Rz= 0.99 R2 0.99 =
,,/ ,,,,)‘” (,,I .” “ 6.”
., ;,,/ +y:
“X’ ,!. ,,,q” ,, I’ “,I ”
300
HS7777 Figure
CS25-M
2. Distribution of Al contents in robot l@s of Al-sensilivr Al-tolemnt corn cultivars grown in nutrient solution with 10.3 A13+ activity a~ pH 4.4 for 24 h (mean ir S.E.. n=3).
200 and MM
loo ” 10
Lower root CEC for tolerant cultivars have also been found by other workers: Kennedy et al. [ 111 for cotton (Goss~~&~ sp.) and Blarney et al. [3] for grassland (Lotus pedunculatus L.). Low root CEC could contribute to Al tolerance when the roots grow into Al toxic conditions [ 111 by excluding polyvalent cations (such as Al), reducing both the binding of Al on exchange sites, and the acidity of the rhizosphere [ 191. The nonexchangeable-Al concentrations in the excised root rips of corn increased linearly with time, and no differences were observed between cultivars (figure 3 C). A similar tendency has been previously demonstrated by Zhang and Taylor [2S, 261 between sensitive and tolerant wheat cultivars. The nonex-
* 30
n 50
C525-M ‘. 70
withal * * 90
DNP = 76.3+1.4xdme -. *. * 110 130 150
RI- 0.99 - ‘. 170
Time (min) Figure 3. Distribution of Al in excised root lips of corn plants (Alsensitive and Al-tolerant cultivars) as B function of time. A, total AI; H, exchangeable-Al; C, nonexchangeable-Al. The Al uptake solution (pH 4.4) was composed of 200 pM CaCl, and 30 pM AICI, with or without 0.1 mM DNP. The treatment was conducted at 25 rt 0.5 “C (mean + S.E.. n=3).
changeable-Al strongly increased for both cultivars when DNP was added @gure 3 C). These results show that cell membranes, under normal metabolic conditions, play an important physiological function against the movement of Al into the cell (symplastic compartment) [21,25]. The higher values of nonexchangeablevol.
36 (6)
1998
466
J. Pintro et al.
Al contents for (325-M than for HS7777 under nonmetabolic conditions, may reflect differences in their cell membranes. Under normal aerobic conditions, the cell membranes of C525-M root tips may be more effective in limiting the entry of Al into the symplastic compartment. Although no differences between cultivars were observed in excised root tips during 180 min of Al uptake figure 3 C), nonexchangeable-Al contents were lower for the tolerant cultivar than for the sensitive cultivar during 24 h of Al uptake (flqurr 2), supporting the suggestion that the tolerance of C525M was based on a mechanism of Al exclusion from root tips. 3. CONCLUSION The elongation of main root of corn plants was reduced by Al in solution. The reduction was marked for the Al-sensitive cultivar (HS7777) compared with the Al tolerant cultivar ((325-M). The Al content in entire roots was not different between cultivars. On the other hand, in the root tips, the Al contents were lowel for (325-M. Although in the presence of DNP in solution the Al contents were higher for C525-M. These results confirm the hypothesis that the tolerance of (325-M appears to be based on a mechanism of exclusion of Al from the root tips. 4. METHODS 4.1. Germination. Corn caryopses (HS7777 single sensitive hybrid and C.525-M single tolerant hybrid) were surface sterilized in I % (v/v) sodium hypochlorite for 30 min, then rinsed with deionized water and germinated on moistened filter paper for 66 h. Seedlings were selected for vigour and uniformity and the length of principal roots was measured. 4.2. Nutrient solution and growth conditions. Seedlings were grown in aerated nutrient solution (7 L) at pH 4.4. The composition of the nutrient solution was based on the analysis of tropical soil extracts [7] and was composed a:3 follows (PM): Ca (200), Mg (IOO), K (400), N-NH: (300), N-NO; (700), S-SO:- (3lS), Zn (0.38), Mn (15). Fe (IO), EDTA (IO), Na (5), P-PO:- (5), Cu (0.16) and MO (0.06). Two Al treatments (Al was added as AICI,~6H,O) and 3 replicates (10 seedlings for each replication) were used. Nominal Al concentrations of 0.0 and 30.0 pM correspond to 0.0 and 10.3 pM Al”+ activity, and to 1.940 and 2.060 pM of ionic strength as calculated by GEOCHEM [ 171. Plants were grown in a growth room at 25 c 0.5 “C and 70 c 1 o/j of relative humidity during 14 h of light period. The photosynthetic photon flux density was 200 pmol&.s-‘. 4.3. Root elongationand determination of total Al. After 24 h seedlings were harvested. The length of the main roots
were remeasured, rinsed once in 200 pM CaCl* (pH 4.4) at 4 f 0S”C [20, 251, and rinsed 3 times with deionized water ( 1 + 0.3 @cm-‘) at 4 f 0.5 “C. The root tips (10 mm) were excised. Root tips include the root cap, meristem and elongation zone [ 161. Main root and root tip samples were dried at 70 “C and weighed. The dry matter was ashed at 500 “C for 2 h and dissolved in 10 mL of 2 % (v/v) HCI into 10 ml test tubes, which were then centrifuged at 1 800 x g for 10 min. Al contents, expressed as mg.kg-’ dry matter, were determined by Inductively Coupled Plasma - ICP (Jobin Yvon JYSOP).
4.4. Determination of nonexchangeable-and exchangeable-Al. Samples of fresh root t$ps were transferred to plastic tubes (50 x 35 mm) perforated at both ends. Nylon sieves of 0.84 mesh.mm-* were placed on both ends held by elastic rings outside the tubes. The plastic tubes were transferred to an aerated solution of 5 mM CaCl, (pH 4.4) at 0 f 0.5 “C for 30 min [20, 2.51. Afterwards, these tubes were rinsed once into 200 J.IM CaCl* (pH 4.4) at 4 + 0.5 “C, rinsed 3 times with deionized water, and then the root tips were prepared for determination of Al by ICP. The exchangeable-Al was calculated by the difference between total Al and nonexchangeable-Al.
4.5. Excising of root tips. After germination,
10 excised root r@s were transferred to each plastic tube. During the time ofexcising of root tips, the plastic tubes (with root tips) were maintained in aerated deionized water at 2.5 f 0.5 “C. When the excised operation was finished, the tubes were transferred to an aerated solution of 200 pM CaCl, (pH 4.4) at 25 * 0.5 “C for 30 min [25]. 4.6. Uptake of Al by excisedroot lips. The beginning of Al uptake by root tips occurred when the plastic tubes were transferred into an aerated solution of 200 pM CaCI, and 30 PM AK& (pH 4.4) at 25 f 0.5 “C. After 0, 5, IO, 15, 20, 30, 60, 90. l-20, 180,240 and 300 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution, rinsed once into a solution of 200 pM CaCl, and rinsed 3 times with deionized water. Afterwards, the root tips were prepared for determination of Al by ICP. 4.7. Determination of nonexchangeableAl. After 10, 30, 60, 120 and 180 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution. The procedure of determination of nonexchangeable-Al of excised root I@ was the same as described above.
4.8. Determination of nonexchangeable-Alin tbe root tips treated with the Al solution containing 2,4-dinitrophenol (DNP). The plastic tubes were removed from the aerated solution of 200 pM CaCl, and were transferred to an aerated solution composed of 200 pM CaCI,, 30 PM AICI, and 0.1 mM DNP (pH 4.4) [251. After 10, 30, 60, 120 and 1X0 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution and prepared for determination of nonexchangeable-Al as described above.
4.9. Determination of CEC of roat tips. Samples of root lips (before Al treatment)
were prepared in order to deter-
ZJptake of Al by the root tips of different
mine the CEC according to the method of Crooke [S]. Samples of dry roots were saturated by 0.01N HCl during 5 min. After, the roots were washed with water to eliminate the excess of HCl in solution. A solution of IN KC1 (pH 7.0) was added and quantities of O.OlN KOH were used to reestablish the pH 7.0. The results were expressed in mmol,kg-’ dry matter.
REFERENCES ] Bennet R.J., Breen CM., The aluminium signal: new dimensions to mechanisms of aluminium tolerance, in: Wright R.J., Baligar V.C, Murrmann RI? (eds.), Plantsoil interactions at low pH, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1991, pp. 703-7 16. ] Bennet R.J., Breen C.M., Fey M.V., The primary site of aluminium injury in the root of Zea mays L.. S. Afr. J. Plant Soil 2 (1985) l-7. [3] Blarney F.P.C., Edwards D.G., Wheeler D.M., Role of root cation-exchange capacity in differential aluminium tolerance of Lotus species, J.-Plant Nutr. 13 (1990) 729744. .] Blarney F.P.C.. Robinson N.J., Asher C.J., Interspecific differences in aluminium tolerance in relation to root cation-exchange capacity, in: Randall R.J., Delhaize E., Richards R.A., Munns R. (eds.), Genetics aspects of plant mineral nutrition, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1993, pp. 91-96. ] Crooke W.M., The measurement of the cation-exchange capacity of plants roots, Plant and Soil 21 (1964) 4349. [6] Delhaize E., Craig S., Beaton C.D., Bennet R.J., Jagadish V.C., Randall P.J., Aluminum tolerance in wheat (Triticum aesrivum L.) I. uptake and distribution of aluminum in roots apices, Plant Physiol. 103 (1993) 685 693. [7] Fallavier P., Babre D., Aventurier A., Bartoli F., Philippy R., Caracterisation et evolution du statut de l’aluminium des sols tropicaux acides. URA Departement Gerdat-Cirad, Montpellier, France, 1988. [S] Foy C.D., Plant adaptation to acid, aluminum-toxic soils, Commun. Soil Sci. Plant Anal. 19 (1988) 959987. [9] Guevara P., Poschenrieder C., Barcelo J., Differential response of four maize (Zea muys L.) varieties to aluminum toxicity, Suelo y Planta 2 (1992) 7 13-72 1. [lo] Huett D.O., Menary R.C., Aluminium uptake by excised roots of cabbage, lettuce, and kikuyu grass, Aust. J. Plant Physiol. 6 (1979) 643-653. [l I] Kennedy C.W., Smith W.C., Ba M.T., Root cuion exchange capacity of cotton cultivars in relation to aluminum toxicity, J. Plant Nutr. 9 (1986) 1 123-l 133. [12] Kinraide T.B., Identity of the rhizotoxic aluminium species, in: Wright R.J., Baligar V.C., Murrmann R.P. (eds.), Plant-soil interactions at low pH. Kluwer Academic Publishers, Dordrecht, The Netherlands, 199 1. pp. 7 17-728.
cultivars
of Zeu mays
467
[ 131 Marschner H., Mechanisms of adaptation of plants to acid soils, in: Wright R.J., Baligar V.C., Murrrnann R.P. (eds.), Plant-soil interactions at low pH, Kluwer Academic Publishers, Dordrecht, The Netherlands, 199 1, pp. 683-702. [ 141 Pavan M.A., Bingham F.T., Toxicity of aluminium to coffee seedlings grown in nutrient solution, Soil Sci. Sot. Am. J. 46 (1982) 993-997. [ 151 Pintro J.C., Barloy J., Fallavier P., Aluminium effects on the growth and mineral composition of corn plants cultivated in nutrient solution at low aluminium activity, J. Plant Nutr. 19 (1996) 729-741. [ 161 Silk W.K., Lord E.M., Eckard K., Growth patterns inferred from anatomical records. Empirical tests using longisections of roots of Zc(l mqvs L., Plant Physiol. 90 ( 1989) 708-7 13. Sposito G., Mattigod S.V., GEOCHEM: A computer program for the calculation of chemical equilibria in soil solution and other natural water systems. University of California, Riverside, LISA, 1980. 118 Tan K., Keltjens W.G., Effects of aluminium on growth, nutrient uptake, proton efflux and phosphorus assimilation of aluminium-tolerant and -sensitive sorghum (Sorghnr~~ &color) genotypes, in: van Beusichem M.L. (ed.), Plant nutrition-physiology and applications, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1990, pp. 397-40 I. 119 1Taylor G.J., The physiology of aluminum tolerance in higher plants, Commun. Soil Sci. Plant Anal. 19 (I 988) 1179-J 194. 1201 Tice K.R., Parker D.R., De Mason D., Operationally defined apoplastic and symplastic aluminum fractions in root tips of aluminum-intoxicated wheat, Plant Physiol. 100 (1992) 309-318. [ 211 Wagatsuma T., Effect of non-metabolic conditions on the uptake of aluminum by plant roots, Soil Sci. Plant I Nutr. 29 (1983) 323-333. [ 221 Wagatsuma T., Characterization of absorption sites for aluminum in the roots, Soil Sci. Plant Nutr. 29 (1983) 499-s 15. [ 231 Wagatsuma T., Akiba R., Low surface negativity of root protoplasts from aluminum tolerance plants species, Soil Sci. Plant Nutr. 35 (1989) 443-452. [ 241 Wagatsuma T., Nakashima T., Tawaraya K., ldentification of aluminium-tolerance protoplasts in the original root protoplast population from several plant species differing in aluminium tolerance, in: Wright R.J., Baligar V.C., Murrmann R.P. (eds.), Plant-soil interactions at low pH, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1991, pp. 789-793. 1251 Zhang G., Taylor G.T., Kinetics of aluminum uptake by excised roots of aluminum-tolerant and aluminum-sensitive cultivars of Tritkutn ac’stivum L., Plant Physiol. 9 1 ( 1989) 1094- 1099. [ 261 Zhang G., Taylor G.T., Kinetics of aluminum uptake in Trihum uestivum L.: identity of the linear phase of aluminum uptake by excised roots of aluminum-tolerant and aluminum-sensitive cultivars, Plant Physiol. 94 ( 1990) 577-584. vol. 36 (6) 1998
Biochern.,
1998, 36 (h), 463-467
Uptake of Aluminium by the root t&s of an Al-sensitive tolerant cultivar of Zea mays Jos6 Intro’*,
and Al-
Jean BarIoy2, Paul FaBavier3
’ Department of Agronomy, State University of Mating& Av Colombo 5790, Maring&PR, 87020-900, Brazil ’ Laboratoire d’agronomie, Ecole nationale sup&ieure agronomique de Rennes, 35042 Rennes, France. ’ Centre de coopCration internationale en recherche agronomique pour le dirveloppement-URA, BP 5035, 34032 Montpellier, France. * Author to whom correspondence should be addressed 1fax 5.5 44 263 42 42) (Received May 2, 1997; accepted November 13, 1997)
Abstract - Uptake and compartmentation of Al in the roots tips were investigated in two corn (Zecr n~,vs L.) cultivars (HS7777 single sensitive hybrid and CS25-M single tolerant hybrid). Seedlings were grown for 24 h in nutrient solution at pH 4.4 characterized by low ionic strength (2,06 FM) and low Al”+ activity (10.3 vM). The root elongation decreased strongly for the sensitive cultivar. There were no differences in the contents of total Al in the entire roots between cultivars. Nonexchangeable-Al and exchangeable-Al contents were higher in the root rips 01‘ HS7777. The sensitive cultivar also showed higher total and exchangeable-Al contents in excised root tips in 180 min of Al uptake. Al uptake for both cultivars increased in solution containing 0. I mM of 2,4-dinitrophenol. Al contents were higher for the tolerant cultivar. 0 Elsevier, Paris Aluminium I Al distribution / Al uptake I dinitrophenol / root tips I Zea mays Al, aluminium /2,4 DNP, 2,4-dinitrophenol / CEC, cation exchangecapacity / ICP, inductively coupled plasma
1. INTRODUCTION Aluminium (Al) toxicity is one of the principal fattors that limits plant growth and production in acid soils [S]. The first visual symptom of Al toxicity is a reduced root growth [ 12, 14, 201 and the toxic effects are more evident in the root tips [ I, 21. The tolerance of plants against Al toxicity is developed using two basic strategies [ 13, 191. The first strategy, blocking the entry of Al into the symplastic compartment could act through a pH barrier at the rhizosphere, a selective permeability of the plasma membrane, the exudation of chelates, or the immobilization of Al in the cell wall. The second strategy, in the symplastic compartment, acts through Al chelation in the cytosol, vacuolar compartmentation, or formation of complexes with Al-binding proteins. The cell wall and the cell membrane (under normal metabolic conditions) probably function as an important barrier to the passive movement of Al into symplastic compartment 1211. Zhang and Taylor 1251 showed under non-metabolic conditions (e.g. 2,4 diniPlant
Physiol. Biochem., 0981-9428/98/06/O Elsevier, Paris
trophenol in uptake solutions) that the Al concentration in wheat (Tuitic~nz aestiwnz L.) root tips was strongly increased . Al may be adsorbed in the cell wall by the negative charges [22]. Even though the root cation exchange capacity (CEC) plays a role in the process of Al tolerance, the results in the literature are contradictory. Lower CEC values were found in the Al tolerant species [ 191. Blarney et al. [4] investigated the Al tolerance in relation to root CEC of 12 plant species (monocots and dicots) and found that tolerance of Al was not closely associated with differences in root CEC values. The selective permeability to Al and negative charges on the surface of the plasma membrane are important characters which determine the passage of Al into the symplasm [23, 241. The negative charges on the surface of the plasma membrane of root cells are stronger in the sensitive plant species. A larger amount of Al is bound to the plasma membrane, and the plasma membrane is eventually destroyed by Al 1231.
464
J. Pintro
et al.
The nonexchangeable-Al can be defined as the Al in the symplastic compartment and the Al precipitated or chelated in the apoplastic compartment, which: cannot be exchanged. The Al in the apoplastic compartment adsorbed on the negative charges of cell wall is defined as exchangeable-Al [20]. The Al toxicity effects on root growth are less correlated with the total Al content in roots [ 131. The analysis of root tips for total Al, although preferable to analysis of whole roots, does not identify differences in Al distribution within root t@~, which may be important in understanding the basis ot’ Al tolerance [6]. The compartmentation of Al in the root tips [ZO, 251 and the comparison of Al uptake by roots under normal and non-metabolic conditions [21) art: bettel parameters. The goal of this study was to investigate Al uptake and AI compartmentation in the root tip.? of an Al-sensitive and Al-tolerant cultivar of corn.
2. RESULTS
AND DISCUSSION
2.1. Root growth The Al treatment (I 0.3 yM Al’+ activity) reduced the elongation of main root for both cultivars, but root growth of C525-M was less inhibited by Al than HS7777 was @gure I). Root growth was reduced to
withoat
Figure 1. Net elongation and C52S-M (Al-tolerant) without or with 10.3 pM n=3).
Al
of the main root of HS7777 (Al-!,ensitive) corn cultivars cultivated in nutriem solution A?+ activity at pH 4.4 for 24 h (mean f S.E..
69 o/ and 43 o/ofor HS7777 and C525-M, respectively, in relation to control. Tan and Keltjens [18] for sorghum (Sorghum bicolor L. Moench), Guevara et al. [9] and Pintro et al. [ 151 for corn (&a mays L.), and Tice et al. [20] for wheat (Tuiri~m aesrivum L.) have found similar differences between Al-sensitive and Al-tolerant cultivars. 2.2. Aluminium
in entire roots and in root tips
There were no differences in the contents of total Al in the entire roots [S69 + 26 mg-kg-’ for HS7777 and 555 f 12 mg.kg-’ for C525-M (mean + S.E., n=3)] between cultivars. However, the Al contents in the root +.s of C525-M were lower than for HS7777 (figure 2). The AI contents for CS2S-M correspond to 55 % of total Al, 2 I % of exchangeable-Al and 73 ‘II, of the nonexchangeable-Al contents found for HS7777. These results (total Al) are in concordance with previous studies 16, 20, 251 working with an At-tolerant and Al-sensitive cultivar of wheat. Total Al content in the entire roots was not a good indicator of the differences in the Al tolerance of the two cultivars. The results showed that the amount of Al in the root tips was an efficient tool for discriminating between the two cultivars. Analyses of Al content in root tips are in agreement with the hypothesis that CS2S-M encodes a tolerance mechanism by exclusion of Al from root tips 161. 2.3. Aluminum
in excised root tips
Total Al contents in excised corn root tips increased with time lfigure 3 A). Uptake of Al was initially higher in the first period (approximately 30 min) for both cultivars. At the end of Al uptake time (300 min), there was a tendency towards an equilibrium value. Total Al concentrations were higher in the HS7777 than in CS2S-M. Similar results in Al-uptake kinetic were found by Huett and Menary [lo] with cabbage (Rmssictr oleracea L.), lettuce (Lnctuca sutiva L.), kikuyu grass (Penrzisetum clundestinium Chiov.) and by Zhang and Taylor [2S ] with wheat, despite differences in experimental conditions (such as species, pH and Al concentrations). Al-uptake kinetic could be described by an exponential function. The exchangeable-Al contents found for the tolerant cultivar were lower than those found for the sensitive cultivar (figtire 3 B). The differences in the exchangeable-Al between cultivars may reflect, in part, the lower root CEC values of root rips for C.525 M (74 f 4 mmol.kg-’ dry matter) than for HS7777 (92 + 3 mmol.kg-’ dry matter) (mean rt S.E., n=3).
Upbake of Al by the root tips of different
Total
-
Al
900 800 700 600 500 400 300 200 100 0
0
l-
of Zeu muys
465
750 700 650 600 550 500 450 400
1100 1000
cultivars
30
60
90
120
150
180 210
240
270
300
330
Exchangeable-Al 250
150 100 HS7117 = 26.2+225.1 50
Nonexchangerbte-Al’
C525-M 10
800
30 .,
.
-
600
-
500
-
70 .,
90
(1 -e(“.m-))
R’ = 0.99
(I-e(-‘mmpo’)
RZ=0.98
110
.,
a,
130 .,
HS7777 with DNP = 86.7+2.8xtime C525-M with DNP 108.3+3.2xtime =
C 700
50 ,
= -17.5+19X3
With
DNP
/? ,,
,zI
400-
..*(-
,,I’
150 .,
170 a,
Rz= 0.99 R2 0.99 =
,,/ ,,,,)‘” (,,I .” “ 6.”
., ;,,/ +y:
“X’ ,!. ,,,q” ,, I’ “,I ”
300
HS7777 Figure
CS25-M
2. Distribution of Al contents in robot l@s of Al-sensilivr Al-tolemnt corn cultivars grown in nutrient solution with 10.3 A13+ activity a~ pH 4.4 for 24 h (mean ir S.E.. n=3).
200 and MM
loo ” 10
Lower root CEC for tolerant cultivars have also been found by other workers: Kennedy et al. [ 111 for cotton (Goss~~&~ sp.) and Blarney et al. [3] for grassland (Lotus pedunculatus L.). Low root CEC could contribute to Al tolerance when the roots grow into Al toxic conditions [ 111 by excluding polyvalent cations (such as Al), reducing both the binding of Al on exchange sites, and the acidity of the rhizosphere [ 191. The nonexchangeable-Al concentrations in the excised root rips of corn increased linearly with time, and no differences were observed between cultivars (figure 3 C). A similar tendency has been previously demonstrated by Zhang and Taylor [2S, 261 between sensitive and tolerant wheat cultivars. The nonex-
* 30
n 50
C525-M ‘. 70
withal * * 90
DNP = 76.3+1.4xdme -. *. * 110 130 150
RI- 0.99 - ‘. 170
Time (min) Figure 3. Distribution of Al in excised root lips of corn plants (Alsensitive and Al-tolerant cultivars) as B function of time. A, total AI; H, exchangeable-Al; C, nonexchangeable-Al. The Al uptake solution (pH 4.4) was composed of 200 pM CaCl, and 30 pM AICI, with or without 0.1 mM DNP. The treatment was conducted at 25 rt 0.5 “C (mean + S.E.. n=3).
changeable-Al strongly increased for both cultivars when DNP was added @gure 3 C). These results show that cell membranes, under normal metabolic conditions, play an important physiological function against the movement of Al into the cell (symplastic compartment) [21,25]. The higher values of nonexchangeablevol.
36 (6)
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J. Pintro et al.
Al contents for (325-M than for HS7777 under nonmetabolic conditions, may reflect differences in their cell membranes. Under normal aerobic conditions, the cell membranes of C525-M root tips may be more effective in limiting the entry of Al into the symplastic compartment. Although no differences between cultivars were observed in excised root tips during 180 min of Al uptake figure 3 C), nonexchangeable-Al contents were lower for the tolerant cultivar than for the sensitive cultivar during 24 h of Al uptake (flqurr 2), supporting the suggestion that the tolerance of C525M was based on a mechanism of Al exclusion from root tips. 3. CONCLUSION The elongation of main root of corn plants was reduced by Al in solution. The reduction was marked for the Al-sensitive cultivar (HS7777) compared with the Al tolerant cultivar ((325-M). The Al content in entire roots was not different between cultivars. On the other hand, in the root tips, the Al contents were lowel for (325-M. Although in the presence of DNP in solution the Al contents were higher for C525-M. These results confirm the hypothesis that the tolerance of (325-M appears to be based on a mechanism of exclusion of Al from the root tips. 4. METHODS 4.1. Germination. Corn caryopses (HS7777 single sensitive hybrid and C.525-M single tolerant hybrid) were surface sterilized in I % (v/v) sodium hypochlorite for 30 min, then rinsed with deionized water and germinated on moistened filter paper for 66 h. Seedlings were selected for vigour and uniformity and the length of principal roots was measured. 4.2. Nutrient solution and growth conditions. Seedlings were grown in aerated nutrient solution (7 L) at pH 4.4. The composition of the nutrient solution was based on the analysis of tropical soil extracts [7] and was composed a:3 follows (PM): Ca (200), Mg (IOO), K (400), N-NH: (300), N-NO; (700), S-SO:- (3lS), Zn (0.38), Mn (15). Fe (IO), EDTA (IO), Na (5), P-PO:- (5), Cu (0.16) and MO (0.06). Two Al treatments (Al was added as AICI,~6H,O) and 3 replicates (10 seedlings for each replication) were used. Nominal Al concentrations of 0.0 and 30.0 pM correspond to 0.0 and 10.3 pM Al”+ activity, and to 1.940 and 2.060 pM of ionic strength as calculated by GEOCHEM [ 171. Plants were grown in a growth room at 25 c 0.5 “C and 70 c 1 o/j of relative humidity during 14 h of light period. The photosynthetic photon flux density was 200 pmol&.s-‘. 4.3. Root elongationand determination of total Al. After 24 h seedlings were harvested. The length of the main roots
were remeasured, rinsed once in 200 pM CaCl* (pH 4.4) at 4 f 0S”C [20, 251, and rinsed 3 times with deionized water ( 1 + 0.3 @cm-‘) at 4 f 0.5 “C. The root tips (10 mm) were excised. Root tips include the root cap, meristem and elongation zone [ 161. Main root and root tip samples were dried at 70 “C and weighed. The dry matter was ashed at 500 “C for 2 h and dissolved in 10 mL of 2 % (v/v) HCI into 10 ml test tubes, which were then centrifuged at 1 800 x g for 10 min. Al contents, expressed as mg.kg-’ dry matter, were determined by Inductively Coupled Plasma - ICP (Jobin Yvon JYSOP).
4.4. Determination of nonexchangeable-and exchangeable-Al. Samples of fresh root t$ps were transferred to plastic tubes (50 x 35 mm) perforated at both ends. Nylon sieves of 0.84 mesh.mm-* were placed on both ends held by elastic rings outside the tubes. The plastic tubes were transferred to an aerated solution of 5 mM CaCl, (pH 4.4) at 0 f 0.5 “C for 30 min [20, 2.51. Afterwards, these tubes were rinsed once into 200 J.IM CaCl* (pH 4.4) at 4 + 0.5 “C, rinsed 3 times with deionized water, and then the root tips were prepared for determination of Al by ICP. The exchangeable-Al was calculated by the difference between total Al and nonexchangeable-Al.
4.5. Excising of root tips. After germination,
10 excised root r@s were transferred to each plastic tube. During the time ofexcising of root tips, the plastic tubes (with root tips) were maintained in aerated deionized water at 2.5 f 0.5 “C. When the excised operation was finished, the tubes were transferred to an aerated solution of 200 pM CaCl, (pH 4.4) at 25 * 0.5 “C for 30 min [25]. 4.6. Uptake of Al by excisedroot lips. The beginning of Al uptake by root tips occurred when the plastic tubes were transferred into an aerated solution of 200 pM CaCI, and 30 PM AK& (pH 4.4) at 25 f 0.5 “C. After 0, 5, IO, 15, 20, 30, 60, 90. l-20, 180,240 and 300 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution, rinsed once into a solution of 200 pM CaCl, and rinsed 3 times with deionized water. Afterwards, the root tips were prepared for determination of Al by ICP. 4.7. Determination of nonexchangeableAl. After 10, 30, 60, 120 and 180 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution. The procedure of determination of nonexchangeable-Al of excised root I@ was the same as described above.
4.8. Determination of nonexchangeable-Alin tbe root tips treated with the Al solution containing 2,4-dinitrophenol (DNP). The plastic tubes were removed from the aerated solution of 200 pM CaCl, and were transferred to an aerated solution composed of 200 pM CaCI,, 30 PM AICI, and 0.1 mM DNP (pH 4.4) [251. After 10, 30, 60, 120 and 1X0 min of Al uptake, 3 plastic tubes were removed from the Al uptake solution and prepared for determination of nonexchangeable-Al as described above.
4.9. Determination of CEC of roat tips. Samples of root lips (before Al treatment)
were prepared in order to deter-
ZJptake of Al by the root tips of different
mine the CEC according to the method of Crooke [S]. Samples of dry roots were saturated by 0.01N HCl during 5 min. After, the roots were washed with water to eliminate the excess of HCl in solution. A solution of IN KC1 (pH 7.0) was added and quantities of O.OlN KOH were used to reestablish the pH 7.0. The results were expressed in mmol,kg-’ dry matter.
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