Universite Catholique de Louvain, Laboratoire de Cytogenetique et Genetique du Developpement, Place Croix du Sud, 5, B-1348 Louvain-la-Neuve, Belgium
Aluminium toxicity is one of the most important factors affecting agricultural productivity and limiting the extension of agriculture throughout the tropics. The effects of Al on plants are numerous. Inhibition of root and stem growth is frequently reported, and symptoms of phosphorus, calcium, iron or magnesium deficiency are also observed on leaves, due to mineral nutrition disturbances. At the cellular level, A1 affects cell walls, membrane lipids, nucleic acids and can induce cell autolysis, as well as calcium deficiency. In 1989, Slaski observed differences in NADkinase activity in two wheat varieties, one A1-tolerant and the other A1-sensitive, in the presence or absence of A1. This enzyme, also called ATP:NAD2'phosphotransferase (E.C.2.7.1.23) is an ubiquitous protein, existing in all organisms, and is at the present time the only known enzyme which catalyses the production of NADP from NAD. The aim of our work was to assay this enzyme activity, in the presence or absence of A1, by using 5 rice (Oryza sativa L.) varieties with different levels of A1 tolerance: A1-sensitive-Aiwu and I Kong Pao, and Al-tolerant-IRAT 104, IRAT 112 and IRAT 140.
Thirty-day-old plants were transferred for 60 days in IRRI nutrient solution (pH5, Yoshida 1976) or in pAl 1500 solution (pH 3.85). The latter culture medium, with unchelated iron, low calcium and phosphorus concentrations, contained per liter: 240.7 mg Of MgSO`4`.7H`2`0, 41.02 mg of Ca(NO`3`)`2`-4H`2`0, 228.6 mg of NH`4`NO`3`, 16.09 mg of KC`l`, 6.16 mg of NaH`2`PO`4`.2H`2`0, 499.98 mg of Al`2`(SO`4`)`3`-18H`2`0, 27.8 mg FeSO`4`.7H`2`0 and microelements according to Yoshida. Fresh root-tip samples. (160 mg per plant) were collected from plants of each variety and from both culture media. They were rinsed twice with demineralized water, ground in liquid N`2` and homogenized in a microgrinder, with 300ul of a solution containing KCl (1 M), EDTA (1 mM), Tris pH 7.5 (50 mM), MgCl`2`.6H`2`0 (2 mM), PVP (2.5% w/v). After addition of 500ul of a solution containing KCl (0.2 M), EDTA (1 mM), Tris pH 7.4 (50 mM), MgCl`2`.6H`2`0 (2 mM), the samples were centrifuged at 15000 g and 4 deg C, for 30 min. The protein content of the supernatants was determined according to Bradford (1976), and the method used to assay NADkinase activity was based on the "enzymatic cycling" procedure of Passonneau and Lowry (1974).
The protein contents of the 5 genotypes were not significantly different on IRRI medium (Table 1). In contrast, on pAl 1500 medium, significant differences were observed between tolerant and sensitive varieties, but not among sensitive genotypes on one hand, and among tolerant ones on the other. The protein content was also affected by the growing medium. It increased in all
Table 1. Protein content (ug prot. mg-1 fresh wt.) of 5 varieties in the
IRRI and pA11500 media (means+\-s.e.)
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Varieties IRRI medium pA11500 medium Comparison LSD between IRRI and pA11500
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1 Kong Pao 0.97+/-0.13 0.20+/-0.02 **
Aiwu 0.96+/-0.34 1.12+/-0.04 NS
IRAT 104 0.98+/-0.18 2.60+/-0.31 **
IRAT 112 1.02+/-0.18 2.55+/-0.35 **
IRAT 140 1.21+/-0.30 2.41+/-0.12 **
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NS, no significant difference; **, significant difference at the 1% level.
tolerant varieties on pAl 1500 as compared to that on IRRI medium. In
contrast, in sensitive varieties, it was either unchanged (Aiwu) or lowered
(I Kong Pao) on the A1 enriched medium. The fact that aluminium is able to
bind with nucleic acids, particularly to DNA, could indirectly explain this
last observation. On the other hand, the increase in protein content
observed in the three IRAT varieties, could be the sign of a higher metabolic
activity allowing the plants to endure the stress. About NADkinase activity
(Table 2), the 5 genotypes were not different when cultured on IRRI medium.
On pAl 1500, the activity of the enzyme was higher in sensitive than in
tolerant varieties, but statistical analysis revealed that only Aiwu was
significantly different from the three IRAT varieties. Comparison of the
activity on the two media showed that it was unchanged in tolerant genotypes
but markedly enhanced in sensitive varieties grown on the A1 enriched
solution, although a significant difference was only recorded for I Kong Pao.
These results were not consistent with Slaski's finding and diverged in two
main points. First, in our case, NADkinase activity in sensitive genotypes
was about twenty times higher on pAl 1500 than on IRRI solution, while it was
unaffected by the culture medium in tolerant varieties. This result
suggested that the increase of NADkinase activity was related to A1
sensitivity rather than to Al tolerance as reported by Slaski. Secondly, the
values we recorded differ enormously from those of Slaski, but are closer
to those reported for colza leaf blades (Maciejewska and Kacperska 1987) or
maize coleoptiles (Dieter and Marme
Table 2. NADkinase activity (pkatals mg-1 prot.) of 5 varieities in the
IRRI and pAl 1500 media (means+/-s.e.)
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Varieties IRRI medium pAl 1500 medium Comparison LSD between IRRI and pAl1500
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1 Kong Pao 0.41+/-0.08 9.67+/-1.22 **
Aiwu 1.02+/-0.47 19.21+/-8.22 NS
IRAT 104 1.28+/-0.95 0.98+/-0.16 NS
IRAT 112 0.97+/-0.29 0.67+/-0.16 NS
IRAT 140 1.92+/-1.11 1.33+/-0.12 NS
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NS, no significant difference; **, significant difference at the 1% level.
1984). Species genotype, plant age and Al treatment duration would be
responsible for this variability, although we do not think that these factors
were sufficient to explain the large divergences observed. Various
mechanisms of A1 tolerance must certainly be involved.It is known that a high NADP/NAD rate (and then a high NADkinase activity) can increase the photosynthetic activity. However, our sensitive genotypes don't seem to possess enough energy to overcome the stress. This characteristic, first postulated by Taylor (1989), is corroborated by the observations that, after two months of culture in pAl 1500 medium, photosynthetic activity, evaluated by the chlorophyll fluorescence method, decreases much more in our two sensitive genotypes than in the three IRAT varieties, and that sensitive plants end up dying when maintained in the presence of the stress factor. On the other hand, we suggest that the tolerant varieties we investigated possess either mechanisms limiting A1 uptake, or mechanisms allowing the detoxification of this element, or finally more or less A1-insensitive metabolic pathways, since NADKinase activity is not enhanced in the presence of A1, while their protein content increases in the presence of A1.
References
Bradford, M. M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.
Dieter, P. and D. Marme, 1984. A Ca2+ , calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane. J. Biological Chemistry 259: 184-189.
Maciejewska, U. and A. Kacperska, 1987. Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants. Physiol. Plant. 69: 687-691.
Passonneau, J. V. and O.H. Lowry, 1974. Nicotinamide-adenine dinucleotides (NAD, NADP, NADH, NADPH): measurement by enzymatic cycling. In Methods of enzymatic analysis, Bergmeyer(ed.), p. 2059-2072.
Slaski, J. J., 1989. Effect of aluminium on calmodulin-dependent and calmodulin-independent NAD kinase activity in wheat (Triticum aestivum L.) root tips. J. Plant Physiol. 133: 696-701.
Taylor, G. J., 1989. Maximum potential growth rate and allocation of respiratory energy as related to stress tolerance in plants. Pl. Physiol. Biochem. 27: 605-611.
Yoshida, S., 1976. Routine procedure for growing rice plants in culture solution. In Laboratory manual for physiological studies of rice, S. Yoshida, D. A. Forno, J. H. Cook and K. A. Gomez (eds.), p. 61-66, I.R.R.I., Manilia, Philippines.