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Indole acetic acid production by Rhizobium: Effect of 2-ketoglutaric acid
0038-0717/82iO20153-03803.0@0 Copyright 0 1982 Pergamon Press Ltd
Sail Bit,/ BhxGm. Vol. 14. pp I53 to 155. 1982 Printed in Great Britain. All rights reserved
INDOLE
ACETIC EFFECT
ACID PRODUCTION OF 2-KETOGLUTARIC
BY RHZZOBZUM: ACID
T. GARCIA-RODRIGUEZ, A. M. GUTIERRIIZ-NAVARRO, R. GARCIA and J. PEREZ SILVA
Departamento
de Microbiologia. Fact&ad de Biologia. Universidad de Sevilla. Sevilla. Spain (ACCqmJd 10 Arrytrst 1981)
acetic acid (IAA) production from tryptophan by cell suspensions of Rhkohium trifdii. R. /rycrminosarum. R. phasroli and R. hcpini was studied in the presence or in the absence of 2-ketoglutaric acid. In R. Iupini, production of IAA was strongly enhanced by the ketoacid. but in fast growing rhizobia it was less enhanced or unaffected. On the other hand, glutamic acid inhibits IAA production by R. melilori, but stimulates IAA production in both R. lquminosarum and R. phusroli. A hypothesis is proposed to explain these results.
Summary---Indole
INTRODUCTION Indole-3-acetic
acid (IAA)
production
from
trypto-
by Rhizobium
has been extensively studied because it is one of the events largely involved in the formation of legume root-nodules (Kefford et al., 1960; Nutman, 1965, 1977). On the basis of their work on the biochemistry and physiology of IAA synthesis, Trinchant and Rigaud (1974) have proposed the most likely biochemical pathway for the oxidation of tryptophan into IAA by rhizobia. Rigaud (1970) and T. Garcia-Rodriguez (unpublished doctoral thesis, University of Seville, 1978) have proved that IAA production can be affected by substances such as 2-ketoglutaric acid (KGA) and glutamic acid (GA) which can act as tryptophan deaminating substrates in the first step of the IAA biosynthetic pathway (Rigaud, 1970; Lantican and Muir, 1967; Truelsen, 1972; Trulfa-Bachi and Cohen, 1973). Werner and Berghaiiser (1976) reported that rhizobial strains vary in their ability to take up KGA or GA. According to them, it is possible to discriminate the fast and slow growing rhizobia on the basis of their uptake kinetics for these substances. Besides this taxonomic implication, the above findings can be used to raise a hypothesis, rhizobial species would be differently affected by KGA and GA in their abilities to oxidise tryptophan to IAA. We show the effect of KGA or GA on IAA production from tryptophan in different Rhizohium strains. phan
MATERIALS AND METHODS
The rhizobial strains used are listed in Tables 3, 2, 5, 4 and 6 for Rhizobium trijidii, R. meliloti, R. phaseoli, R. leguminosarum and R. lupini respectively. All strains were shown to be infective on their specific hosts by nodulation tests. The bacteria were grown in a mannitol-yeast extract-salts medium (Vincent, 1970). The cultures were incubated at 28°C. on a gyratory shaker at 120 rev min-‘. To study IAA production, cells from cultures in logarithmic phase were harvested by centrifugation at
10,OOOgfor 10min. washed twice with 0.1 M phosphate buffer (pH, 7.5) and resuspended in the same buffer to a final absorbance at 600nm equal to 0.5 (about 6 x lo* cells ml-‘). Tryptophan was added to the cell suspensions to provide a concentration of 1 mM, using a concentrated solution sterilized by filtration through a millipore filter (0.45 pm pore dia). After 48 h at 28°C. the IAA was measured by the method of Gutitrrez-Navarro et al. (1979). For comparative studies incubations were carried out in the absence and in the presence of KGA or GA at the concentrations indicated in each experiment. All assays were performed by triplicate, and statistical estimations of the errors of extraction and assay methods indicated that differences in IAA production greater than 12% are significant. RESULTS AND DlSCUSSlON
In order to know whether the presence of KGA exerts any influence on IAA production by rhizobia, cell suspensions from R. trifolii RS-90, R. meliloti RS-94, R. leguminosarum RS-288, R. phasroli RS-92 and R. lupini RS-110 were incubated in the presence
of several concentrations of KGA. and their ability to transform tryptophan into IAA were determined. The results (Table 1) show that IAA production was significantly affected in all rhizobial strains by KGA at some of the concentrations used, the R. lupini RS-110 being the more strongly affected. However, R. leguminosarum RS-288 was not affected when the KGA concentration was equal or lower than 1 mM. Furthermore, the effect of 1 mtu KGA on IAA production was studied using five different strains of R. meliloti (Table 2), R. trifolii (Table 3) and R. leguminosarum (Table 4); four strains of R. phaseoli (Table 5) and three strains of R. lupini (Table 6). The results are consistent with those reported above and permit us to conclude that R. lupini is the species that has the highest sensitivity to KGA. while in R. leguminosarum, the strain RS-285 was the only one significantly affected by KGA (Table 4). The observations can be correlated with the different rates of KGA uptake shown by these rhizobial species. Werner and Bergatlser (1976) reported that R.
153
154
T. GAKCIA-RODKIGIJ / PI I!/. Table
I. Indole
acetlc
centrations
of I-ketoglutaric
0 Strains R. rrijdii RS-90 R. phasroli RS-92 R. leguminosarum RS-288 R. melilori RS-94 R. lupini RS-110
Concentration of KGA (mM) 0.1 1 5 (pg IAA ml-‘)
1.40 1.35 1.00 3.31 0.43
Table 2. Indole acetic acid (IAA) production from tryptophan by cell suspensions of five strains of R. mr/ilofi in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
Strains RS-94 RS-96 RS-138 RS-168 RS-208
IAA production (ilg ml-‘) with KGA without KGA 3.31 3.35 5.30 1.77 1.16
4.53 5.07 5.94 2.39 2.16
Effects’ 1.36 1.51 1.12 1.35 1.86
’ Expressed as the ratio: IAA produced in the presence of KGAjlAA produced in the absence of KGA. Table 3. Indole acetic acid (IAA)
production from tryptoof five strains of R. tr[folii in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
phan by cell suspensions
Strains RS-55 RS-90 RS-91 RS-169 RS-170
IAA production (ccgml- ‘) without KGA with KGA 3.98 1.48 3.11 2.59 0.95
5.79 4.06 5.45 6.46 5.36
‘) from tryptophan by cell in the presence of several conacid (KGA)
(pg ml
acid production
suspensions of different strains of Rhimhium
Etfects’ 1.60 2.74 1.75 2.40 5.50
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
4.9 2.3 1 1.03 3.65 1.23
10.36 2.18 1.00 4.53 5.57
Strains RS-284 RS-285 RS-286 RS-287 RS-288
IAA production (pg ml- ‘) with KGA without KGA 2.36 3.50 4.13 1.61 1.00
2.39 4.50 4.32 1.80 1.00
Effects’ 1.01 1.28 1.04 1.11 1.00
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA. kguminosarum shows lower rate of KGA uptake than R. trijblii and R. lupini do, and the slow growing rhizobia (such as R. lupini) assimilate KGA from the medium more intensively than the faster growing species.
24.64 17.90 13.36 3.36 9.00
The action of glutamic acid (a substance which can also act as deaminating agent of tryptophan) (TruffaBachi and Cohen, 1973) on IAA production by fast growing rhizobia was also studied. R. trijblii RS-169, R. meliloti RS-94, R. lrgrrminosarrrm RS-288 and R. phtrsroli RS-92 were incubated in the absence or presence of GA at various concentrations, and their abilities to produce IAA were measured. The results of such experiments (Table 7) show that GA, at all of the concentrations assayed, enhanced IAA production by R. phasroli RS-92 and R. leguminosarlrm RS-288. R. trifolii RS-169 is not affected save at the concentration of 10 mM of GA, and IAA production by R. mdiloti RS-94 is significantly inhibited at all concentrations of GA assayed. The results suggest that R. legrrminosarum would preferentially use glutamic acid to deaminate tryptophan. since its IAA production is weakly affected by the KGA (Table 4) and strongly affected by GA (Table 7). This fact is also consistent with the rate of glutamic acid uptake found by Werner and Berghaiiser (1976), who report that R. kgrrminosarrrm takes up glutamic acid more rapidly than KGA.
Table 5. lndole acetic acid (IAA) production from tryptophan by cell suspensions of four strains of R. phascwli in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
Strains Table 4. lndole acetic acid (IAA) production from tryptophan by cell suspensions of five strains of R. /cgur?linosarunI in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
21.70 15.70 4.39 3.58 7.10
10
RS-92 RS-278 RS-280 RS-281
IAA production (pg ml- ‘) without KGA with KGA 1.30 0.62 1.02 1.64
2.38 0.77 1.64 5.18
Eflects’ 1.83 1.24 1.60 3.15
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
Table 6. lndole acetic acid (IAA) production from tryptophan by cell suspensions of three strains of R. lupini in the presence or absence of 1 mM 2-ketoglutaric acid (KGA) Strains RS-100 RS-108 RS-110
IAA production (pg ml- I) without KGA with KGA 0.75 0.63 0.46
9.08 3.77 5.59
Effects’ 12.11 5.99 12.15
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
Indolc acetic acid pro ‘duction by Rhi~bittm Table 7. Indole acetic acid (IAA) production
from tryptophan by cell suspensions of different strains of ~bizobiu~ in the presence of several concentrations of glutamic acid (GA)
acid.
Anales
Ayrarias
dei
&win),
155 institifto
Srrir:
~a~~~~nul de i~~e.~ti#u~iones
Generu~ 6,
159-167.
KEFFOKDN. P.. BROCKWELLJ. and ZWAR J. A. (1960)
The symbiotic synthesis of auxin by legumes and nodule bacteria and its role in nodule development.
Strains
R. trifolii RS-169 R. phaseoii RS-92 R. leguminosarum RS-288 R. meliloti RS-94
Concentration of GA (mM) 10 0 0.1 1 (pg IAA ml-‘: 1.82
1.86
1.62
1.78
2.16
0.31
0.38
0.34
0.69
0.54
0.96
1.62
1.27
1.70
2.20
4.61
4.03
3.69
2.01
1.12
On the other hand, GA inhibits IAA production by R. meliioti RS-94 (Table 7), which can be explained assuming that R. meliloti does not use this amino acid
as a deaminating substrate of tryptophan, and having in mind that GA is a product of the mediated KGA tryptophan deamination (Truelsen, 1972) one can expect that GA exerts, principally at higher concentrations a significant effect on the equilibrium of the first reaction of the pathway for IAA synthesis in this organism. This agrees well with the results of Table 1, which indicate that IAA production by R. mefiioti is significantly affected by 1 mM KGA, but not at higher concentrations.
REFERENCES GUTIERREZ-NAVARRO A. M.. BELLOCINR. A.. GARCIA-R•D-
KIGUEZT.. PARRASL. and TORRESA. (1979) An assay method for indole acetic acid in presence of indole lactic
Journal
of Biologicul
Austruliun
Science 43, 456-467.
L~NTICANB. P. and MUIR R. M. (1967) Isolation and properties of the enzyme system forming indoleacetic acid. Hunt ,Physiolo#p 42, 1158-l 160. NUTMANP. S. (1965) The relation between nodule bacteria and the legume host in the rhizosphere and in the process of infection. In Eco/oav of Soil-Borne Plan? Pathoyens (K. F. Baker and W.-e. Snyder, Eds). pp. 231-247. University of California Press, Berkeley. NUTMAN P. S. (1977) Study frameworks for symbiotic nitrogen fixation. In Recent Deve~opmencs in Nitr~~gen Fixation (W. Newton, J. R. Postgate and C. RodriguezBarrueco, Ed& pp. 433447. Academic Press, London. RIGAUDJ. (1970) L.‘acide indolyl-3-lactique et son m&tabolisme chez Rhi:obium. Archiv fiir Mikrobiologie 72, 297-307.
TRENCHANT J. C. and RIGAUDJ. (1974) Lactate dehydrogenase from Rhizobium. Purification and role in indole metabolism. Physiologia Pluntorum 32, 394-399. T~UELSENT. A. (1972) Indole-3-ovruvic acid as an intermediate in the conversion of-tryptophan to indole3-acetic acid. I. Some characteristics of tryptophan transaminase from mung bean seedlings. Physioiogia Plantarum
26,289-295.
TRUFFA-BACHIP. and COHEN G. N. (1973) Amino acid metabolism. Annual Review oJ ~io~~ernjstr~ 42, 113134. VINCENTJ. M. (1970) A ~flrnlal~r the Practical Study of Roor-~~~~~e Bocferia. IBP Handbook 15. Blackwell, Oxford. WERNER D. and BERGHA~~SER K. (1976) Discrimination of Rhizobium japonicum. Rhizohium lupini, Rhizohium trifolii, Rhizobium leguminosarum and of bacteroids by uptake of 2-ketoglutaric acid, glutamic acid and phosphate. Archives of Microhio/o#y 107, 257-262.
Sail Bit,/ BhxGm. Vol. 14. pp I53 to 155. 1982 Printed in Great Britain. All rights reserved
INDOLE
ACETIC EFFECT
ACID PRODUCTION OF 2-KETOGLUTARIC
BY RHZZOBZUM: ACID
T. GARCIA-RODRIGUEZ, A. M. GUTIERRIIZ-NAVARRO, R. GARCIA and J. PEREZ SILVA
Departamento
de Microbiologia. Fact&ad de Biologia. Universidad de Sevilla. Sevilla. Spain (ACCqmJd 10 Arrytrst 1981)
acetic acid (IAA) production from tryptophan by cell suspensions of Rhkohium trifdii. R. /rycrminosarum. R. phasroli and R. hcpini was studied in the presence or in the absence of 2-ketoglutaric acid. In R. Iupini, production of IAA was strongly enhanced by the ketoacid. but in fast growing rhizobia it was less enhanced or unaffected. On the other hand, glutamic acid inhibits IAA production by R. melilori, but stimulates IAA production in both R. lquminosarum and R. phusroli. A hypothesis is proposed to explain these results.
Summary---Indole
INTRODUCTION Indole-3-acetic
acid (IAA)
production
from
trypto-
by Rhizobium
has been extensively studied because it is one of the events largely involved in the formation of legume root-nodules (Kefford et al., 1960; Nutman, 1965, 1977). On the basis of their work on the biochemistry and physiology of IAA synthesis, Trinchant and Rigaud (1974) have proposed the most likely biochemical pathway for the oxidation of tryptophan into IAA by rhizobia. Rigaud (1970) and T. Garcia-Rodriguez (unpublished doctoral thesis, University of Seville, 1978) have proved that IAA production can be affected by substances such as 2-ketoglutaric acid (KGA) and glutamic acid (GA) which can act as tryptophan deaminating substrates in the first step of the IAA biosynthetic pathway (Rigaud, 1970; Lantican and Muir, 1967; Truelsen, 1972; Trulfa-Bachi and Cohen, 1973). Werner and Berghaiiser (1976) reported that rhizobial strains vary in their ability to take up KGA or GA. According to them, it is possible to discriminate the fast and slow growing rhizobia on the basis of their uptake kinetics for these substances. Besides this taxonomic implication, the above findings can be used to raise a hypothesis, rhizobial species would be differently affected by KGA and GA in their abilities to oxidise tryptophan to IAA. We show the effect of KGA or GA on IAA production from tryptophan in different Rhizohium strains. phan
MATERIALS AND METHODS
The rhizobial strains used are listed in Tables 3, 2, 5, 4 and 6 for Rhizobium trijidii, R. meliloti, R. phaseoli, R. leguminosarum and R. lupini respectively. All strains were shown to be infective on their specific hosts by nodulation tests. The bacteria were grown in a mannitol-yeast extract-salts medium (Vincent, 1970). The cultures were incubated at 28°C. on a gyratory shaker at 120 rev min-‘. To study IAA production, cells from cultures in logarithmic phase were harvested by centrifugation at
10,OOOgfor 10min. washed twice with 0.1 M phosphate buffer (pH, 7.5) and resuspended in the same buffer to a final absorbance at 600nm equal to 0.5 (about 6 x lo* cells ml-‘). Tryptophan was added to the cell suspensions to provide a concentration of 1 mM, using a concentrated solution sterilized by filtration through a millipore filter (0.45 pm pore dia). After 48 h at 28°C. the IAA was measured by the method of Gutitrrez-Navarro et al. (1979). For comparative studies incubations were carried out in the absence and in the presence of KGA or GA at the concentrations indicated in each experiment. All assays were performed by triplicate, and statistical estimations of the errors of extraction and assay methods indicated that differences in IAA production greater than 12% are significant. RESULTS AND DlSCUSSlON
In order to know whether the presence of KGA exerts any influence on IAA production by rhizobia, cell suspensions from R. trifolii RS-90, R. meliloti RS-94, R. leguminosarum RS-288, R. phasroli RS-92 and R. lupini RS-110 were incubated in the presence
of several concentrations of KGA. and their ability to transform tryptophan into IAA were determined. The results (Table 1) show that IAA production was significantly affected in all rhizobial strains by KGA at some of the concentrations used, the R. lupini RS-110 being the more strongly affected. However, R. leguminosarum RS-288 was not affected when the KGA concentration was equal or lower than 1 mM. Furthermore, the effect of 1 mtu KGA on IAA production was studied using five different strains of R. meliloti (Table 2), R. trifolii (Table 3) and R. leguminosarum (Table 4); four strains of R. phaseoli (Table 5) and three strains of R. lupini (Table 6). The results are consistent with those reported above and permit us to conclude that R. lupini is the species that has the highest sensitivity to KGA. while in R. leguminosarum, the strain RS-285 was the only one significantly affected by KGA (Table 4). The observations can be correlated with the different rates of KGA uptake shown by these rhizobial species. Werner and Bergatlser (1976) reported that R.
153
154
T. GAKCIA-RODKIGIJ / PI I!/. Table
I. Indole
acetlc
centrations
of I-ketoglutaric
0 Strains R. rrijdii RS-90 R. phasroli RS-92 R. leguminosarum RS-288 R. melilori RS-94 R. lupini RS-110
Concentration of KGA (mM) 0.1 1 5 (pg IAA ml-‘)
1.40 1.35 1.00 3.31 0.43
Table 2. Indole acetic acid (IAA) production from tryptophan by cell suspensions of five strains of R. mr/ilofi in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
Strains RS-94 RS-96 RS-138 RS-168 RS-208
IAA production (ilg ml-‘) with KGA without KGA 3.31 3.35 5.30 1.77 1.16
4.53 5.07 5.94 2.39 2.16
Effects’ 1.36 1.51 1.12 1.35 1.86
’ Expressed as the ratio: IAA produced in the presence of KGAjlAA produced in the absence of KGA. Table 3. Indole acetic acid (IAA)
production from tryptoof five strains of R. tr[folii in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
phan by cell suspensions
Strains RS-55 RS-90 RS-91 RS-169 RS-170
IAA production (ccgml- ‘) without KGA with KGA 3.98 1.48 3.11 2.59 0.95
5.79 4.06 5.45 6.46 5.36
‘) from tryptophan by cell in the presence of several conacid (KGA)
(pg ml
acid production
suspensions of different strains of Rhimhium
Etfects’ 1.60 2.74 1.75 2.40 5.50
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
4.9 2.3 1 1.03 3.65 1.23
10.36 2.18 1.00 4.53 5.57
Strains RS-284 RS-285 RS-286 RS-287 RS-288
IAA production (pg ml- ‘) with KGA without KGA 2.36 3.50 4.13 1.61 1.00
2.39 4.50 4.32 1.80 1.00
Effects’ 1.01 1.28 1.04 1.11 1.00
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA. kguminosarum shows lower rate of KGA uptake than R. trijblii and R. lupini do, and the slow growing rhizobia (such as R. lupini) assimilate KGA from the medium more intensively than the faster growing species.
24.64 17.90 13.36 3.36 9.00
The action of glutamic acid (a substance which can also act as deaminating agent of tryptophan) (TruffaBachi and Cohen, 1973) on IAA production by fast growing rhizobia was also studied. R. trijblii RS-169, R. meliloti RS-94, R. lrgrrminosarrrm RS-288 and R. phtrsroli RS-92 were incubated in the absence or presence of GA at various concentrations, and their abilities to produce IAA were measured. The results of such experiments (Table 7) show that GA, at all of the concentrations assayed, enhanced IAA production by R. phasroli RS-92 and R. leguminosarlrm RS-288. R. trifolii RS-169 is not affected save at the concentration of 10 mM of GA, and IAA production by R. mdiloti RS-94 is significantly inhibited at all concentrations of GA assayed. The results suggest that R. legrrminosarum would preferentially use glutamic acid to deaminate tryptophan. since its IAA production is weakly affected by the KGA (Table 4) and strongly affected by GA (Table 7). This fact is also consistent with the rate of glutamic acid uptake found by Werner and Berghaiiser (1976), who report that R. kgrrminosarrrm takes up glutamic acid more rapidly than KGA.
Table 5. lndole acetic acid (IAA) production from tryptophan by cell suspensions of four strains of R. phascwli in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
Strains Table 4. lndole acetic acid (IAA) production from tryptophan by cell suspensions of five strains of R. /cgur?linosarunI in the presence or absence of 1 mM 2-ketoglutaric acid (KGA)
21.70 15.70 4.39 3.58 7.10
10
RS-92 RS-278 RS-280 RS-281
IAA production (pg ml- ‘) without KGA with KGA 1.30 0.62 1.02 1.64
2.38 0.77 1.64 5.18
Eflects’ 1.83 1.24 1.60 3.15
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
Table 6. lndole acetic acid (IAA) production from tryptophan by cell suspensions of three strains of R. lupini in the presence or absence of 1 mM 2-ketoglutaric acid (KGA) Strains RS-100 RS-108 RS-110
IAA production (pg ml- I) without KGA with KGA 0.75 0.63 0.46
9.08 3.77 5.59
Effects’ 12.11 5.99 12.15
’ Expressed as the ratio: IAA produced in the presence of KGA/lAA produced in the absence of KGA.
Indolc acetic acid pro ‘duction by Rhi~bittm Table 7. Indole acetic acid (IAA) production
from tryptophan by cell suspensions of different strains of ~bizobiu~ in the presence of several concentrations of glutamic acid (GA)
acid.
Anales
Ayrarias
dei
&win),
155 institifto
Srrir:
~a~~~~nul de i~~e.~ti#u~iones
Generu~ 6,
159-167.
KEFFOKDN. P.. BROCKWELLJ. and ZWAR J. A. (1960)
The symbiotic synthesis of auxin by legumes and nodule bacteria and its role in nodule development.
Strains
R. trifolii RS-169 R. phaseoii RS-92 R. leguminosarum RS-288 R. meliloti RS-94
Concentration of GA (mM) 10 0 0.1 1 (pg IAA ml-‘: 1.82
1.86
1.62
1.78
2.16
0.31
0.38
0.34
0.69
0.54
0.96
1.62
1.27
1.70
2.20
4.61
4.03
3.69
2.01
1.12
On the other hand, GA inhibits IAA production by R. meliioti RS-94 (Table 7), which can be explained assuming that R. meliloti does not use this amino acid
as a deaminating substrate of tryptophan, and having in mind that GA is a product of the mediated KGA tryptophan deamination (Truelsen, 1972) one can expect that GA exerts, principally at higher concentrations a significant effect on the equilibrium of the first reaction of the pathway for IAA synthesis in this organism. This agrees well with the results of Table 1, which indicate that IAA production by R. mefiioti is significantly affected by 1 mM KGA, but not at higher concentrations.
REFERENCES GUTIERREZ-NAVARRO A. M.. BELLOCINR. A.. GARCIA-R•D-
KIGUEZT.. PARRASL. and TORRESA. (1979) An assay method for indole acetic acid in presence of indole lactic
Journal
of Biologicul
Austruliun
Science 43, 456-467.
L~NTICANB. P. and MUIR R. M. (1967) Isolation and properties of the enzyme system forming indoleacetic acid. Hunt ,Physiolo#p 42, 1158-l 160. NUTMANP. S. (1965) The relation between nodule bacteria and the legume host in the rhizosphere and in the process of infection. In Eco/oav of Soil-Borne Plan? Pathoyens (K. F. Baker and W.-e. Snyder, Eds). pp. 231-247. University of California Press, Berkeley. NUTMAN P. S. (1977) Study frameworks for symbiotic nitrogen fixation. In Recent Deve~opmencs in Nitr~~gen Fixation (W. Newton, J. R. Postgate and C. RodriguezBarrueco, Ed& pp. 433447. Academic Press, London. RIGAUDJ. (1970) L.‘acide indolyl-3-lactique et son m&tabolisme chez Rhi:obium. Archiv fiir Mikrobiologie 72, 297-307.
TRENCHANT J. C. and RIGAUDJ. (1974) Lactate dehydrogenase from Rhizobium. Purification and role in indole metabolism. Physiologia Pluntorum 32, 394-399. T~UELSENT. A. (1972) Indole-3-ovruvic acid as an intermediate in the conversion of-tryptophan to indole3-acetic acid. I. Some characteristics of tryptophan transaminase from mung bean seedlings. Physioiogia Plantarum
26,289-295.
TRUFFA-BACHIP. and COHEN G. N. (1973) Amino acid metabolism. Annual Review oJ ~io~~ernjstr~ 42, 113134. VINCENTJ. M. (1970) A ~flrnlal~r the Practical Study of Roor-~~~~~e Bocferia. IBP Handbook 15. Blackwell, Oxford. WERNER D. and BERGHA~~SER K. (1976) Discrimination of Rhizobium japonicum. Rhizohium lupini, Rhizohium trifolii, Rhizobium leguminosarum and of bacteroids by uptake of 2-ketoglutaric acid, glutamic acid and phosphate. Archives of Microhio/o#y 107, 257-262.