Genotypic Differences in Aluminium Tolerance of Soybean (Glycine max L.) as Affected by Ammonium and Nitratenitrogen Nutrition

J.PlantPhysiol. Vol. 132.pp. 702-707(1988)

Genotypic Differences in Aluminium Tolerance of Soybean (Glycine max L.) as Affected by Ammonium and NitrateNitrogen Nutrition F.

KLOTZ

and W. J.

HORST

Institut fUr Pflanzenernahrung, Universitat Hohenheim, Postfach 700562, D-7000 Stuttgart 70, Federal Republic of Germany Present address: Institut fiir Pflanzenernahrung, Universitat Hannover, Herrenhauserstr. 2, D-3000 Hannover 21, Federal Republic of Germany Received October 2, 1987 . Accepted January 20, 1988

Summary Soybean (Glycine max L.) genotypes differed considerably in response of root elongation to aluminium supply in solution culture. Root elongation was more sensitive to Al than nitrate and ammonium net uptake rates, which were higher in Al treated plants. Nitrate reductase activity, which was also higher in Al treated plants in more basal root zones, was unaffected on a much higher activity level in the apical 2 mm root zone. Genotypic differences in Al tolerance were not related to differences in NH4 + versus N0 3 - uptake, nitrate reductase activity and related changes of pH in the rhizosphere. This was confirmed by a sand culture experiment comparing NH4 + and N0 3 - nutrition and direct measurement of pH at the root surface along the roots.

Key words: Glycine max, Aluminium, Soybean, Nitrogen form, Nitrate reductase activity, Genotypic differences. Abbreviations: NRA

=

nitrate reductase activity.

Introduction Plant species, as well as genotypes within species may differ widely in tolerance to high Al supply of the nutrient medium (Foy, 1983). There is increasing interest by plant breeders in exploiting this genetic variability for the breeding of cultivars adapted to acid soils with high Al supply (BoyeGoni and Marcarian, 1985; Aniol, 1984 a). There are examples of successful selection of plants adapted to acid soils on the basis of screening for Al tolerance in solution culture, especially for grasses (Reid et aI., 1971; Spain et aI., 1975). However, in soybean we did not find any correlation between Al tolerance under controlled conditions and under field conditions (unpublished results). This might be due to differences in ionic composition of nutrient and soil solutions affecting Al tolerance of the plants. The N form, which in most nutrient solutions is predominantly N0 3 - but © 1988 by Gustav Fischer Verlag, Stuttgart

NH4 + in most acid soils, could be especially important. Since the activity of toxic Al species in solution is dependent on pH, preferential uptake and reduction of N0 3 - leading to higher pH is expected to increase Al tolerance of plants, which in fact is indicated by the results of Fleming (1983), and Taylor and Foy (1985). Also, accumulation of organic acids in the symplast as a consequence of N0 3 - reduction may increase Al tolerance of the roots because of inactivation of Al by complexation (Suhayda and Haug, 1985). However, other authors did not find any effect of N source (Wagatsuma and Yamasaku, 1985) or even increased Al tolerance of plants in the presence of NH4 + compared to N0 3 - (Rorison, 1985; Van Praag et aI., 1985; Klotz and Horst, 1987). The objective of the present study was to clarify the role of the N form and pH changes at the root surface due to differences in N0 3 - and NH4 + uptake and assimilation on Al tolerance of soybean genotypes.

Aluminium tolerance of soybean

Material and Methods

703

Both experiments were conducted with 4 replicates per treatment (3 plants per replicate).

Growing conditions ofplants Plants were grown in a growth chamber at 30°/25 °C day/night temperature, 16 h daylength, 150 Wm -2 light intensity, and 70 % relative humidity. Seeds were germinated for 2 days between filter paper soaked with 1 mM CaS04; subsequently seedlings were transferred to 221 plastic vessels. The following nutrient solutions with different N forms have been used (J.tM/I): N03 - solution: KN0 3 750; Mg(N0 3h 325; NH4N03 solution: KC1750; MgCh 325; NH4 N0 3 700; NH4 + solution: KC1750; MgCb 325; NH4 CI1400; and CaS04 250; FeEDDHA 20; KH2P0 4 10; H 3B0 3 8; CUS04 0.2; ZnS04 0.2; MnS04 0.2; (NH4)6M07024 0.2. Al was added as AICh. The pH of the nutrient solution was adjusted (4.2 in water culture, 4.0 in sand culture) and kept constant with 0.01 N HCl or KOH by automatic titration (in water culture). Screening experiment for Al tolerance 11 soybean genotypes germinated for 2 days in 1 mM CaS04 were grown in 221 plastic vessels containing nutrient solution with N as N0 3 -. Al (74.1 J.tM) was added to half of the plants. Each treatment was repeated 6 times (2 plants per replicate). After 4 days, plants were harvested for determination of dry weights, lengths of primary root and the longest secondary root. N uptake experiment Plants were precultured for 4 days in NH 4N0 3 - solution (2211 pot) without AI. Then Al (56 J.tM) was added to half of the plants. 14 hours after beginning of the Al treatment 10 plants per replicate (3) were transferred into aerated uptake solution (250 ml) with the same nutrient composition as during preculture but with reduced N concentration (100 J.tM N as NH4N0 3). N uptake was calculated from N depletion of the uptake solution. NH4 + concentrations were determined colorimetrically by the method of Nelson (1983); N0 3 concentrations were measured by ion chromatography (Dionex 2000i/SP).

Nitrate reductase experiments 1. Plants were precultured in nutrient solution with N as NH4N0 3, without AI. After 3 days Al (56 J.tM) was added to half of the plants. 13 hours after beginning Al treatment, secondary root tips of the plants (1 cm) were excised, weighed and transferred into the incubation medium. 4 days after beginning Al treatment the same procedure was repeated with the rest of the plants. 2. Plants were pre cultured as described above before Al (56 J.tM) was added to half of the plants. 6 hours after beginning Al treatment, secondary roots of plants were cut into segments with varying distances from the root tip (0-2, 2-10, 10-15mm), weighed and transferred into the incubation medium. Nitrate reductase activity (NRA) in root tissues (at least 50 mg fresh weight per sample) was determined after the method of Hunter et al. (1982). Depending on the experimental purpose, N0 3 - (100 mmolll) was added to the incubation medium or not. Nitrate reduction in the incubation medium was stopped by adding 2 ml HCI (25 % v/v) + sulfanilamide (1 % w/w) to the incubation medium (2 ml). Subsequently an aliquot (1 ml) was mixed with 0.5 ml of N-naphtylethylenediaminedihydrochlorid solution (0.02 % w/w); N0 2- was then measured colorimetrically at 546nm.

Culture substrate experiment After germination, half of the plants were grown in water culture as described above. The rest were transferred into 31 plastic pots filled with quartz sand (grain diameter: approximately 1 mm). The pots were leached two times per day with 255 ml kg -1 sand (corresponding to 1x water capacity) of fresh nutrient solution of the same composition as in solution culture (s.a.). Each culture substrate treatment included 3 N forms (N0 3 - , NH4 N0 3 , NH4 +) and 2 Al treatments (0, 74.1 J.tM Al in water culture; 0, 741 J.tM Al in sand culture). The experiment was conducted with 3 replicates and 4 plants per replicate. After 4 days plants were harvested for determination of total root length. Roots were washed free of sand, spread on a transparency film (see Fig. 1) and total root length measured with a graphical analyzer (Digiplan Am 02). pH measurement at the root surface Plants were grown in minirhizotrones filled with quartz sand, 3 replicates per treatment. The pots were leached as described above two times per day with fresh nutrient solution, containing N as NH4N0 3 and 741 J.tM AI. After 3 days the plexiglas covers were taken off and the sand was infiltrated with agar containing the pH indicator bromocressol green (0.006 %) after the method of Marschner and Romheld (1983). The pH at the root surface was then measured using a microelectrode as described by Haussling et al. (1985). The experiment included three replicates.

Results A range of soybean genotypes were grown in solution culture with and without Al supply (Table 1). After 4 days of Al treatment dry matter production of shoot and roots was hardly affected. However, root elongation, especially of secondary roots, was much more sensitive to AI. The product of the length of the primary root and the longest secondary root, which is highly significantly correlated to total root length (not shown), clearly indicate differences between the genotypes in response to AI. This is illustrated in Fig. 1 showing 2 genotypes with contrasting Al tolerance. These 2 genotypes were used for further studies with the objective to get a better understanding of the reasons for differential response of genotypes to AI. Al tolerance could be due to higher nitrate compared to ammonium uptake and assimilation leading to an increase in pH, thus inactivating Al in the apoplast. Therefore, in a first step, net uptake of N0 3 - and NH4 + from a solution containing N as NH4N0 3 was studied. After a preculture period of 4 days without Al followed by 14 h Al treatment, N0 3 - and NH4 + depletion of the solution was monitored in the presence and absence of AI. Whereas N0 3 - uptake rates (Fig.2) steadily declined with time due to decreasing concentration in solution, NH4 + uptake showed a biphasic uptake pattern indicating non-equilibrium conditions. N0 3 - and NH4 + uptake-rates were comparable. N0 3 - as well as NH4 + uptake-rates were higher in Al treated plants. The genotypes did not differ in N4 + uptake, but clear differences were apparent in N0 3 - uptake. Whereas N0 3 -

704

F.

KLOTZ

and W. J.

HORST

Table 1: Shoot and root growth of soybean genotypes as affected by aluminium (74.1 pM). Duration of aluminium treatment: 4 days. root length

genotypes

shoot dry weight (mg/plant) -AI +AI

root dry weight (mg/plant) -AI +AI

primary root (cm) +AI -Al

longest secondary root (cm) -AI +AI

Mc Call Ronda Caloria Fiskeby Europa Gieso Gamba Aura Maple Arrow Effi Sito

34.7 39.6 37.1 38.2 42.9 35.5 52.6 46.4 44.6 42.3 54.4

17.9 24.0 22.2 19.6 29.1 27.7 27.2 30.5 21.3 24.0 23.5

23.2 27.4 26.2 26.1 29.1 25.7 29.3 22.5 28.1 28.5 31.2

2.8 5.1 5.3 3.7 7.7 4.0 5.1 5.7 5.0 5.5 6.7

LSD 0,05

Ronda

36.1 38.1 34.0 30.8 36.8 38.2 45.5 44.5 37.9 35.2 43.7 9.5

Maple Arrow

20.2 24.1 19.6 16.6 27.7 19.3 27.6 27.3 20.0 20.0 20.4

20.1 27.0 24.4 20.3 29.9 23.3 25.9 17.9 21.6 19.0 24.4

2.7

5.4

Ronda

3.1 4.1 3.8 2.6 4.7 2.7 3.6 3.2 2.5 2.3 2.4 1.4

primary root x longest secondary root (+AI in % of -AI) 84.3 78.5 66.4 63.2 61.2 61.0 60.3 45.3 36.2 28.4 28.3 26.3

Maple Arrow

J l

)

~----------AI --------~

~-------+AI --------~

uptake was higher for the AI-sensitive genotype Maple Arrow in the absence of AI, N0 3 - uptake in presence of Al increased to a much higher level in the AI-tolerant genotype Ronda. Not only N0 3 - uptake but especially N0 3 - reduction in the roots could lead to a pH increase in the apoplast. Therefore NRA was measured in root tips (1 cm) of plants differing in age and duration of Al treatment (Fig. 3). Independent upon pre culture and analytical conditions, NRA was comparable for both genotypes in control plants (- AI); however, there was a tendency for decreasing NRA in Al treated roots of the AI-tolerant genotype Ronda and increasing NRA in the AI-sensitive genotype Maple Arrow. Since cell division is restricted to the apical 2 mm and cell elongation to 10 mm (data not shown), NRA of these different zones of the root tips were compared (Fig. 4). In order to relate NRA to short term inhibition of root elongation by AI, NRA was measured after 6 h of Al treatment. The 2 mm root apex showed a nearly 10 times higher NRA than older root segments. No differences could be observed between

Fig. 1: Effect of aluminium (56/LM) on root and shoot morphology of two soybean genotypes. Duration of Al treatment: 4 days.

the genotypes in the absence of AI. Al tended to decrease NRA in the 2 mm root apex of genotype Ronda more and to increase NRA in the basal non-elongating root zones less than in genotype Maple Arrow. If regulation of pH in the apoplast and at the root surface is important for Al tolerance, considerable differences in Al tolerance should occur between plants supplied with either N0 3 - or NH4 + -N. These differences should be especially clear in sand culture compared to solution culture with pH control. Therefore both genotypes were grown in solution and in sand culture using the same composition of the nutrient solution (Fig. 5). However, in sand culture Al concentration had to be increased by a factor of 10 in order to get a comparable inhibition of root elongation by AI. Independent of N form and substrate, root elongation of the genotype Ronda was less depressed by Al compared to Maple Arrow, confirming the higher Al tolerance of Ronda. In no case did NH4 + nutrition increase Al sensitivity compared to N0 3 nutrition. On the contrary, in the presence of NH4 + plants appeared less sensitive to AI.

Aluminium tolerance of soybean Rondo

plant age 3 d duration of AI treatment. 6 h

Maple Arrow

NHZ uptake-rate

NH: uptake-rate

[nM em-I root length]

705

NOj added to ineubot,on medium

[nM em-I root length]

18

NRA

[nM NOi g-1 fresh wI. h- 1)

14

Rondo

440

10 6

360

2

280 200

NOj uptake -rate

NOj uptake-rote

[nM em-I root length]

[nM em-I root length]

18 .AI

14 .0-'

10

,0-'-

y=5.76.5.20 Inx

.AI 0 y =1.81.5.19 Inx ''---

,D'-

•

6

120

0

I

•

•

40

I

3

4

5

6 [h]

0-2

NRA

[mm)

[nM NOi g-1 fresh wt.

-AI

Mople Arrow

440

2 2

O-AI • • AI

2

3

4

5

360

6 [h]

Duration of uptake period

280

Fig. 2: Effect of aluminium (56 ttM) on ammonium and nitrate net uptake-rates of 2 soybean genotypes calculated on the basis of NH4 + and N0 3 - depletion of a nutrient solution containing lOOttM N as NH4N0 3 • Preculture for 4 days without AI, then 14h with Al at 1400 ttM as NH4N03. Bars indicate LSD o.05 .

200 120

40

Prediction of pH changes in the rhizosphere in sand culture were verified by direct measurement of pH using microelectrodes at the root surface of plants grown for 3 days in the presence of Al and NH 4N0 3 as the N source (Fig. 6)_ Although the pH of the bulk soil had been adjusted to pH 4.0, the pH at the root surface, especially at the root apices, was much higher, making inactivation of Al most likely. This could explain the much higher Al tolerance of the plants in sand culture compared to solution culture. However, no differences existed between the genotypes_

plont age duration of AI treotment

0-2

NRA

Fig.3: Effect of aluminium (56 ttM) on nitrate reductase activity in root tips (1 cm) of 2 soybean genotypes as affected by plant age, duration of Al treatment and addition of N0 3- to the incubation medium. N supply 1400 ttM as NH4N0 3 • Bars indicate S.D.

Discussion The existence of considerable genetic variability in Al tolerance in the generally Al sensitive plant species Glycine

4d 13 h

ptont age:

8d

duration of AI treatment 4 d NOj added to incubation medilJm no NOj added to incubation med.um

NRA [nM NO;: g-' fresh wI. h")

NRA [nM NO,- g" fresh wI. h")

160

160

160

140

140

140

120

120

120

100

100

100

80

80

80

60

60

60

40

40

40

20

20

20

Rondo

Mople

Arrow

[mm]

Fig. 4: Effect of aluminium (56 ttM) on nitrate reductase activity in root-tip segments of 2 soybean genotypes. N supply 1400 ttM N as NH4N0 3 . Bars indicate S.D.

NOj added to incubation medium

[nM NO, g" fresh wI. h-')

10-1S

2 - 10 root tip s~ment

Ronda

Mople

Arrow

o

-AI • • AI (56~MI

I

Rondo

I Mopl. Arrow

F. KLOTZ and W. J. HORST

706

reI. root length (-AI =100)

Ell Rondo •

Mople Arrow

I

LSOo.o.S

I

LSOo..o.S

N03 NH4N03 NH4 solution culture 74.1 IJM Al

N03

Fig. 5: Inhibition by aluminium of total root length of 2 soybean genotypes as affected by N form and culture substrate. Duration of Al treatment: 4 days.

NH4N03 NH4 sond culture 741IJM Al

Maple Arrow

Ronda

5.2

5.0

5.5

5.0

4.2 4.1

4.6 4.8 20. mm" 5.3 10. ..

4.5

S.3 S.3

4.6 20 mm"

5.5 10 .. 5.4 5 .. 5.0

It

distance from apex

max L. could be confirmed (Tablel, Devine et aI., 1979; Sapra et aI., 1982). Inhibition of root elongations, especially of the secondary roots, was a most sensitive parameter for Al injury. Root elongation was much more sensitive to Al than N0 3 - and NH4 + uptake (Fig. 1) and N0 3 - reduction in growing root tips (Fig. 2 and 3), indicating the principle toxic effect of Al in the root apoplast. Contrary to the observations of Jarvis and Hatch (1986) with white clover, N0 3 uptake rate was even higher in AI-treated plants. A higher N0 3 - uptake per unit root length could compensate for reduced total root length, due to a comparable N requirement of the shoot which was hardly, affected by Al in dry weight accumulation (Table 1). Since N uptake of the total root system was studied, inhibition of N uptake of the growing sites of the root can not be excluded. However, there was no clear effect of Al on NRA at these sites neither after short term (Fig.3) or longer term (Fig.2) Al treatment. On the contrary, there was a tendency of even increased NRA in the AI-sensitive genotype, an observation which was also made by Foy and Fleming (1982) working with wheat genotypes. The distribution of NRA along the roots clearly shows the necessity of concentrating on root tips when effects of Al on root elongation and physiological parameters are related.

S"

Fig. 6: pH in the bulk soil and at the root surface of 2 soybean genotypes grown for 3 days in sand culture, leached 2 x per day with nutrient solution containing 1400 JtM N as NH4N0 3 and 741 JtM AI.

There were no clear differences in N uptake and N0 3 - reduction between the genotypes in the absence of AI. Differences in the presence of Al were the consequence rather than the reason for growth reduction by AI. It is therefore unlikely that the differences between the soybean genotypes in Al tolerance are due to differences in NH4 + versus N0 3 uptake or N0 3- reduction as has been claimed for other plant species (Mugwira and Patel, 1977; Fleming, 1983; Taylor and Foy, 1985). According to these authors preferential uptake of N0 3- and higher N0 3 - reduction will induce an increase in pH in the rhizosphere, thus inactivating AI. In order to check the importance of pH control in the rhizosphere for Al tolerance, a sand culture experiment with different N forms was conducted (Fig. 5). Although it can be assumed that NH4 + nutrition led to pH decrease and N0 3 nutrition to pH increase in the rhizosphere (Marschner and Romheld, 1983), no differences in Al tolerance were observed in relation to the N source. A comparable or even higher Al tolerance in the presence of NH4 +, in spite of lower pH, could be due to blocking of Al binding sites in the apoplast (Klotz and Horst, 1987) or to a shift in Al speciation in solution to less toxic species (Alva et al., 1986). However, as indicated by Fig. 6 for N~N03 - grown plants and by

Aluminium tolerance of soybean

HiuBling et al. (1985), plants may control the pH at the root tips independent of the N form supplied. The maintenance of a high pH around the root tips in sand culture could explain the much higher Al tolerance of the plants compared to solution culture. Independent of N form and culture substrate, however, the differences in Al tolerance between the genotypes remained the same. This has also been reported by Wagatsuma and Yamasaku (1985) comparing barley genotypes. It can be concluded, that control of pH at the root surface is of minor importance for genotypical Al tolerance in soybean. Detoxification of AI, either within the symplast (Aniol, 1984 b) or in the apoplast by complexion of Al through root exudates (Horst et al., 1982) is probably more important. Acknowledgements We thank Mrs. B. Abraham, E. Gorgus and B. Seeber for skilful technical assistance, Dr. D. L. Godbold, University of Gottingen, for critical reading of the manuscript, and the Commission of the European Communities for financial support.

References ALVA, A. K.,D. G. EDWARDS, C. J. ASHER, and F . P . C. BLAMEY: Relationships between root length of soybean and calculated activities of aluminum monomerers in nutrient solution. Soil Sci. Soc Am. J. 50, 959-962 (1986). ANIOL, A .: Introduction of aluminum tolerance into aluminum sensitive wheat cultivars. Z. Pflanzenziicht. 93, 331- 339 (1984 a). - Induction of aluminum tolerance in wheat seedlings by low doses of aluminium in the nutrient solution. Plant Physiol. 76, 551-555 (1984 b). BOYE-GONI, S. R. and V. MARCARIAN: Diallel analysis of aluminum tolerance in selected lines of grain sorghum. Crop Sci. 25, 749-752 (1985). DEVINE, T. E., C. D . Foy, D. L. MASON, and A. L. FLEMING: Aluminum tolerance in soybean germ plasm. Soybean genetics Newsletter 6 (1979). FLEMING, A. L.: Ammonium uptake by wheat varieties differing in Al tolerance. Agron. J. 75,726-730 (1983). Foy, C. D .: The physiology of plant adaption to mineral stress. Iowa State J. Res. 57, 355-391 (1983). Foy, C. D . and A. L. FLEMING: Aluminum tolerances of two wheat genotypes related to nitrate reductase activities. J. PI. Nutr. 5, 1313 - 1333 (1982). HXUSSLlNG, M., E. LEISEN, H. MARSCHNER, and V. ROMHELD: An improved method fo r non-destructive measurements of the pH

707

at the root-soil interface (rhizosphere). J. Plant. Physio!. 117, 371-375 (1985). HORST, W . J., A. WAGNER, and H . MARSCHNER: Mucilage protects root meristem from aluminium injury. Z. Pflanzenphysio!. 105, 435-444 (1982). HUNTER, W. J., c. J. FAHRlNG, S. R. OLSEN, and L. K. PORTER: Location of nitrate reduction in different soybean cultivars. Crop Sci. 22,944-948 (1982). JARVIS, S. C. and D . J. HATCH: The effects of low concentrations of aluminium on the growth and uptake of nitrate-N by white clover. Plant and Soil 95, 43-55 (1986). KLOTZ, F. and W. J. HORST: Effect of ammonium and nitratenitrogen nutrition on aluminium tolerance of soybean (Glycine max L.) Submitted to Plant and Soil (1987). MARSCHNER, H . and V. ROMHELD: In vivo measurement of root-induced pH changes at the soil-root interface: effect of plant species and nitrogen source. Z. Pflanzenphysio!. 111,241-251 (1983). MUGWIRA, L. M. and S. U. PATEL: Root zone pH changes and ion uptake imbalances by triticale, wheat and rye. Agron. J. 69, 719-722 (1977). NELSON, D. W.: Determination of ammonium in KCl extracts of soils by the salicylate method. Commun. Soil Sci. PI. Anal. 14, 1051-1062 (1983). REID, D . A., A. L. FLEMING, and C. D . Foy: A method for determining aluminum response of barley in nutrient solution in comparison to response in AI-toxic soil. Agron. J. 63, 600-603 (1971). RORISON, L H .: Nitrogen source and the tolerance of Deschampsia jlexuosa, Holcus lanatus and Bromus erectus to aluminium during seedling growth. J. Ecol. 73, 83 - 90 (1985). SAPRA, V. T., T. MEBRATHU, and L. M . MUGWlRA: Soybean germplasm and cultivar aluminium tolerance in nutrient solution and bladen clay loam soil. Agron. J. 74, 687 -690 (1982). SPAIN, J. M., R. H. HOWELER, and F. CALVO: Differential species and varietal tolerances to soil acidity in tropical crops and pastures. In: BOURNEMISZA, E. and A. ALVARADO {eds.}: Soil management in tropical America. University Consortium on Soils of the Tropics, Soil Science Dept., N.C. State University, Raleigh, 313-335 (1975). SUHAYDA, C. G . AND A. HAUG: Organic acids reduce aluminum toxicity in maize root membranes. Phys. Plant. 68, 189-195 (1986). TAYLOR, G. J. and C. D. Foy: Mechanisms of aluminium tolerance in Triticum aestivum (wheat). IV. The role of ammonium and nitrate nutrition. Can. J. Bot. 63, 2181-2186 (1985). VAN PRAAG, H. J., F. WEISSEN, S. SOUGNEZ-REMY, and G. CARLETTI: Aluminium effects on spruce and beech seedlings II. Statistical analysis of sand culture experiments. Plant and Soil, 83, 339-356 (1985). WAGATSUMA, T. and K. YAMASAKU: Relationship between differential aluminum tolerance and plant induced pH change of medium among barley cultivars. Soil Sci. PI. Nutr. 31, 521-535 (1985).