Academic Open Internet Journal ISSN 1311-4360

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Volume 17, 2006

CONTENT OF SUPEROXIDE ANION-RADICAL AND ACTIVITY OF SUPEROXIDE DISMUTASE IN PEA SEEDLINGS WITH DIFFERENT NODULATION ABILITY UNDER INOCULATION BY NODULAR BACTERIA

Galina G. Vasilieva, Anatoly К. Glyanko, Nina V. Mironova, Tat’yana Е. Putilina

Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of the Russian Academy of Science

e-mail – ustaft@sifibr.irk.ru

ABSTRACT. The research was focused on the level of superoxide anion-radical (О₂^.-) and activity of superoxide dismutase (SOD) in pea (Pisum sativum L.) seedlings   with normal (Marat variety) and disturbed (pea mutants: non-nodular and super-nodular) by nodulation process upon their inoculation by nitrogen-fixing bacteria  (Rhizobium leguminosarum bv. viceae, strain CIAM 1026). There were distinguished differences in О₂^.- level and SOD activity in the seedlings with various nodulation ability, depending on their inoculation by rhizobia. О₂^.- and SOD are supposed to be involved in protective and regulatory mechanisms of the host-plant. 

Key words:  Pisum sativum L., Rhizobium leguminosarum, superoxide anion-radical, superoxide dismutase

Formation of legume-rhizobial symbiosis is accompanied by the change of level of active oxygen forms (AOF), such as superoxide anion-radical (О₂^.-) and hydrogen peroxide (Н₂О₂). Nevertheless, physiological role of AOF at the initial stages of symbiotic interaction remains unclear. The available literary data are often contradictory. Thus, a number of authors believe that AOF formation is associated with the perception by the plant of a specific rhizobial Nod-factor and is necessary for nodulation. For example, AOF are formed in the process of nodulation in Sesbania rostrata [1]. Recognition of compatible Nod-factor Sinorhizobium meliloti rapidly induces in the host-plant space localized AOF formation in root hairs  Medicago truncatula [2, 3]. Mutants S. meliloti, forming Nod-factor with altered structure, and non-nodulating plant mutant, whose ability to form AOF is equally weakened. At the same time there are data, which confirm that Nod-factors stimulate oxidation burst in order to block the induction of nodulation plant marker genes in case of incompatible interaction  [4]. Electronic microscopic research of alfalfa roots infected with S. meliloti, demonstrated local formation of AOF in the infection threads matrix, which surrounds bacteria, but not in the bacteria themselves [4, 5]. Mutants S. meliloti, which do not synthesize  superoxide dismutase, changed their symbiotic properties intensely: they induced weak nodulation in alfalfa and development of defect bacteroids [6]. These data show that rhizobia, which penetrated into the root, are under control of the host-plant with AOF participating in this control. However, such kinds of work are very few and have been conducted on singular species of legumes. 

Using rhizobia strains and pea plants, which were “changed” by this or that symbiotic feature, is one of the ways to learn about physiological mechanisms of legumes-rhizobial interaction. Pea mutants with various nodulation ability (ability to form nodules), are a convenient model for investigation of regulatory and protective mechanisms of the host-plant at the initial stages of legume-rhizobial symbiosis. That is why we set out to study the level of О₂^.- and SOD activity in pea mutants with disturbed nodulation: К14а ("non-nodular") and Nod3 ("super-nodular") in comparison with Marat variety characterized by normal nodulation.

MATERIAL AND METHODS

The initial material was presented by 48-hours old pea seedlings (Marat variety, selection by the Tulun state selection station, Russia), as well as mutant К14а, which does not form nodules (“non-nodular”) and mutant Nod3, which forms excessive amount of nodules (“super-nodular”). The seedlings were grown in cuvets on wet filter paper in thermostat at  22 ^оС. 48-hours old seedlings were placed in special chambers in a vertical position so that inoculate would not get on the seedlings’ epicotyls. Chambers for the seedlings were plastic rectangular tanks with the dimensions  12 х 8 х 6 cm, split in tow parts by a horizontal partition with apertures with the diameter slightly less than the pea seed diameter. Seedlings roots via the apertures in the partition were placed in the lower part of the chamber on wet filter paper, whereas seeds and epicotyls were located in the top part.

Roots of the initial seedlings were inoculated by bacteria Rhizobium leguminosarum bv. viceae (production strain CIAM 1026) in the concentration 2 х 10⁷ cells of ml ^-1 suspension based on 1 ml  root ^–1. Strain CIAM 1026 was received from the collection  of the All-Russian scientific-research institute of agricultural microbiology ( Saint-Petersburg, Russia).  This strain is characterized by  high efficiency of nitrogen-fixing ability for different pea varieties and forms  [7].

After inoculation the seedlings were further grown at 22 ^оС during 2 days. Non-inoculated seedlings of the same age served as control.

Determination of О₂^.- level was based on its ability to reduce cytochrome  с (cyt. с) [8]. That is why О₂^.- level was evaluated based on cytochrome с reductase activity (cyt. с RА) of seedlings’ roots and epicotyls, which prior to determination were washed by running water, then by distilled water and dried by filter paper.  Roots and epicotyls’ segments 1.0-1.5 mm long were placed in microcuvets with 0.01 М potassium-phosphate buffer (рН 7.4), which contains 20 μМ of cytochrome с (“Aldrich”, США), 10 mМ of sodium azide (NaN₃), 0.1 mМ EDTA, 50 mМ NaCl. The total volume of buffer solution in microcuvet is 4 ml. With the view to determine cytochrome c reduction, reaction solution (diffusate) was sampled 1 hour after the incubation and its optical density was measured at  λ = 410 nm on spectrophotometer SF-56 ("LOMO", Russia) in microcuvets 1 cm long.  Based on the difference extinction co-efficient between oxidized and reduced forms of cyt. с (Є = 2.21 ^. 10 ⁴ М ^–1 cm ^-1), there was calculated cyt. с RA of roots and epicotyls and expressed in nmol of reduced cyt. с h^-1 g^-1 FM. SOD impact on cyt. с reduction by pea seedlings segments was determined by the addition of SOD (“ICN”, USA) (100 μg ml ^-1) to the reaction mixture, into which NaN₃ was not introduced.

SOD activity was determined by the method based on its ability to inhibit hydroxilaminchloride oxidation reaction with the formation of nitrite in the presence of О₂^.-  generators (system xanthine : xanthine oxidase) [9]. The plant material was grind in the cold in porcelain mortars in 65 mМ phosphate buffer (рН 7.8). Homogenate was centrifuged at lowered temperature within 30 min. at 20000 g. Then there was performed dialysis of supernatant against buffer (within 21 hour at +2, +4 ^оС) to release from low-molecular anti-oxidants. Total protein content in dialyzed supernatant was determined using amide-black dye [10].

The reaction mixture with the total volume of 2 ml consisted of distilled water (0.5 ml), 65 mМ of phosphate buffer (рН 7.8) (1 ml), xanthine oxidase (“Sigma”, USA) – 0.3 ml of enzyme suspension containing 60 μg of protein; 1.5 μM of xanthine solution ("ICN", USA) (0,1 ml) and 1 μМ of hydroxilaminchloride water solution (0.1 ml). The reaction started from the addition of xanthine oxidase and was carried out in water-bath at  25 ^оС within 20 min.

To determine nitrite as a product of hydroxilaminchloride oxidation, 0.5 ml of 0.33% sulphanilic acid and 0.5 ml of 0.1% α-naftilamin solution (“Serva”, Germany) were added to 0.5 ml of the above reaction mixture. The acquired mixture was shaken and left for coloring at room temperature. 20 minutes after optical density was measured on spectrophotometer   at λ = 530 нм.

Addition to the reaction mixture of plant  dialyzed extract instead of water inhibited oxidation of hydroxilaminchloride and nitrite formation, which confirmed presence of SOD, whose activity was calculated on the basis of inhibition degree of the given reaction.  For the sake of calculation there was constructed a calibrating curve via addition to the reaction mixture of different amounts of SOD. The enzyme activity was expressed in the activity units (Е) per 1 mg of protein. 1 unit of SOD activity corresponds to 2 μg of protein and causes complete inhibition of nitrite formation from hydroxilaminchloride. 

The tables present arithmetic means of 3-5 tests and identify standard error (М±Е). Reliability of differences was assessed by Student’s criterion.

RESULTS AND DISCUSSION

The study showed that after inoculation by rhizobia О₂^.- level in pea (Marat variety) roots inconsiderably changed after 24 hours (increase by  20 % is unreliable) and dropped by 23 % after 48 hours (reliably at Р = 0.95, ν = 4). In the roots of mutant Nod3 there was observed decrease of this AOF after 24 and 48 hours after inoculation respectively by 21 and 31 % (reliably at Р ≥ 0.95, ν = 4). Mutant К14а showed an insignificant change in О₂^.- level after 24 hours (reduction by 10 % is unreliable), it increased by 33 % after 48 hours (reliably at Р > 0.95, ν = 4) (Table 1).

SOD activity 24 hours after inoculation by rhizobia considerably decreased in the roots of mutant К14а (by 75%) and Marat variety – by 52 %  (reliably at Р > 0.99, ν = 4). 48 hours after inoculation SOD activity in the roots of these pea genotypes increased as compared to the first day, nevertheless it was still lower than in control: in Marat variety – by 40 % (reliably at Р > 0.95, ν = 4), in mutant  К14а it did not significantly differ from control (reduction by 17 % is unreliable). In the roots of mutant Nod3 SOD activity after inoculation changed insignificantly (Table 2).

Comparison of О₂^.- level in pea roots after their inoculation by rhizobia with SOD activity shows that change of this AOF level is not always connected with relevant change of SOD activity. Thus, 24 hours after inoculation of Marat variety roots their SOD activity reduced by 2.1 times, the increase of О₂^.- level was insignificant (Table 1). After inoculation of the roots of mutant К14а enzyme activity reduced by 4 times, whereas reduction of О₂^.- level was also insignificant. At the same time, reduction of О₂^.- level in the roots of Marat variety after 48 hours was observed along with simultaneous reduction of SOD activity. These results confirm that nodular bacteria cause change in О₂^.- level not only at the expense of SOD activity change, but at the expense of its generation with participation of various mechanisms.

The character of О₂^.- level change as well as earlier found decrease of  Н₂О₂  content [11] in Marat variety pea roots after inoculation are apparently associated with the suppression of host-plant protection system by rhizobial Nod-factor [ 12, 13 ], which contributes to root infecting by rhizobia. Nevertheless, this does not exclude the ability of legume to control infection process via AOF local formation in the infection thread after penetration of certain amount of bacteria, as it was discovered by electronic microscopic investigations in the course of alfalfa interaction with  S. meliloti [4, 5].

Reduction of О₂^.- level and simultaneous decrease of Н₂О₂ content [11] in the roots of mutant Nod3 after inoculation is apparently associated with the disturbance of mechanisms controlling infection process, which leads to the increase of rhizobia penetration in the root tissues.

The character of О₂^.- level change with the simultaneous increase of Н₂О₂ content [11] in the roots of mutant К14а 24 hours after inoculation,  is, vice versa, likely to be explained by the inclusion of protective mechanisms excluding rhizobia penetration in the root of this “non-nodular” mutant. Inability of this mutant to form nodules does not seem to point to its lack of response to rhizobia, which may be regarded in this case as standard pathogens, whose adhesion and penetration in the root tissues of which is accompanied by the activation of plant protective system.

Different character of AOF level the roots of pea genotypes varying in the nodulation ability, after inoculation by rhizobia, shows their interaction into regulatory  and protective mechanisms  at the initial stages of legume-rhizobial interaction.

Inoculation of roots by rhizobia caused different change in О₂^.- level in epicotyls of the pea genotypes studied (Table 1). The Marat variety demonstrated increase of О₂^.- level in epicotyls 24 and 48 hours after inoculation by 36% and 66% respectively (reliably at Р ≥ 0.95, ν = 4). In epicotyls of mutant К14а the level of this AOF decreased by 30% 24 hours after inoculation (reliably at Р > 0.95, ν = 4) and increased by 65 %  after 48 hours (reliably at Р > 0.99, ν = 4). The character of О₂^.- level change in the roots and epicotyls of this mutant was of the same character. The mutant Nod3 demonstrated decrease of О₂^.- level in epicotyls, as well as in the roots, 24 and 48 hours after inoculation, by 58% and 67 % respectively (reliably at Р > 0.99, ν = 4).

After roots inoculation by nodular bacteria SOD activity was observed to reduce in epicotyls of all the investigated pea genotypes. It was particularly significant in the mutant Nod3 (Table 2). The change of О₂^.- level in epicotyls of the given pea types is likely to be due to the processes of its generation and not dismutation.

Therefore, after inoculation of pea roots by nodular bacteria the level of  О₂^.- and SOD activity in epicotyls changes, though this organ does not participate in infecting, nodulation and nitrogen-fixation. The change of level of the given AOF in epicotyl may be conditioned by systemic signaling of the plant, whose mechanisms are currently insufficiently studied. The increase of О₂^.- level in epicotyls after inoculation of pea roots with normal nodulation (Marat variety), as well as increase of H₂О₂  content [11] may be a part of induced protective reactions, which prevent rhizobia infection of this organ.

Reduction of О₂^.- level in epicotyls of the pea mutant Nod3 after roots inoculation is apparently connected with the disturbance of infecting and nodulation processes regulation in the roots and weakening of protective mechanisms in the whole seedling.

The character of change of О₂^.- level in epicotyls of the mutant К14а after inoculation of the roots is likely to be explained by the lack of infection of the roots of this mutant.

The results of the studies show that pea plants characterized by different nodulation ability also vary in the character of the change of О₂^.- level and SOD activity both in the roots and epicotyls, which speaks in favor of their involvement in the mechanisms, which determine nodulation ability of the legume.  The results acquired confirm that in the course of formation of symbiotic relations between nodular bacteria and pea plants superoxide anion-radical and superoxide dismutase are involved in control for roots infecting and in protective reactions, which exclude infection of epicotyl – an organ that does not take part in nodulation.

                        ACKNOWLEDGMENTS

The authors thank prof. K.K. Sidorova (Institute of Cytology and Genetics, SD RAS, Novosibirsk, Russia) for kindly providing a pea mutants.

Literature cited

1. D'Haeze W., Mathis R., De Rycke M., Holsters M. // Abstract Book 13^th International N₂ Fixation Congress.   McMaster University Press. Toronto, 2001. No L59,  P. 19.

2. Ramu S.K., Peng H.M., Cook D.R. // Abstr. Int. Soc. for Mol. Plant-Microbe Interac., APS Press, St. Paul, MN, 1999,  P. 71.

3. Ramu S.K., Peng H.M., Cook D.R. // Mol. Plant-Microbe Interac. 2002, V. 15,  P. 522-528.

4.  Herouart D., Baudouin E., Frendo P. et al. // Plant Physiol. Biochem.  2002,    V. 40,  P. 619-624.

     5. Santos R., Herouart D., Sigaud S. et al. // Mol. Plant-Microbe Interac. 2001, V. 14,  P. 86-89.

6. Santos R., Herouart D., Puppo A., Touati D. // Mol. Microbiol.  2000, V. 38,  P.   750-759.

7. Safronova V.I., Novikova N.I. // J. Microbiol. Methods.  1996, V. 24, P. 231-237

8. Doke N. // Physiol. Plant Pathol.  1983,  V. 23,  P. 345-357.

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10. Buzun G.A., Dzhemukhadze К.М., Mileshko L.F. // Plant Physiology.  1982, V. 29, P. 198-200  (In Russian ).

  11. Vasilieva G.G., Glyanko А.К., Mironova N.V. // Agricultural biology. 2004, № 3, P. 101-105 ( In Russian).

  12. Bueno P., Soto M.J., Rodrigues-Rosales M.P. et al. // New Phytol.  2001, V. 152, P. 91-99.

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Table 1. Cytochrome с reductase activity (cyt. с RА) of pea seedlings with different nodulation ability and impact of inoculation by nodular bacteria Rhizobium leguminosarum bv. viceae (strain CIAM 1026)

Seedlings age, day	Control (without inoculation)		Inoculation R, leguminosarum
			Inoculate impact time, day	цит. с RA
	cyt. с RA  (nmol h ^–1 g^-1 FM)			nmol h ^–1 g ^–1 FM		% to control
	root	epicotyl		root	epicotyl	root	epicotyl
Marat variety
2	36.5 ± 2.8
3	21.6 ± 2.0	62.1 ± 5.1	1	26.0 ± 1.3	84.6 ± 6.3	120	136
4	12.3 ± 1.0	28.6 ± 2.7	2	9.5 ± 0.2	47.6 ± 4,5	77	166
Mutant К14а ("non-nodular")
2	11.5 ± 0.8	5.1 ± 0.5
3	15.8 ± 1.3	8.4 ± 0.7	1	13.7 ± 1.0	5.9 ± 0.5	90	70
4	14.9 ± 1.0	11.2 ± 0.1	2	19.8 ± 1.2	18.5 ± 0.6	133	165
Mutant Nod3 ("super-nodular")
2	12.0 ± 0.10	12.3 ± 0.6
3	20.8 ± 1.2	14.9 ± 1.5	1	16.4 ± 0.6	6.3 ± 0.5	79	42
4	17.5 ± 1.9	14.9 ± 1.0	2	12.1 ± 0.3	4.9 ± 0.3	69	33

Table 2. SOD activity in pea seedlings with various nodulation ability depending on inoculation by nodular bacteria  Rhizobium leguminosarum  bv. viceae (strain CIAM 1026)

Seedlings age, day	Control (without inoculation)		Inoculation by R. leguminosarum
			Inoculate impact time, day	SOD activity
	SOD activity  (Е mg ^–1protein)			Е mg ^–1 protein		% to control
	root	epicotyl		root	epicotyl	root	epicotyl
Marat variety
2	1.4 ± 0.07	0.8 ± 0.01
3	2.7 ± 0.17	1.3 ± 0.13	1	1.3 ± 0.12	1.0 ± 0.10	48	77
4	2.5 ± 0.16	1.6 ± 0.02	2	1.5 ± 0.05	1.5 ± 0.09	60	94
Mutant К14а ("non-nodular")
2	0.4 ± 0.04	0.6 ± 0.02
3	1.2 ± 0.10	0.5 ± 0.03	1	0.3 ± 0.03	0.4 ± 0.03	25	80
4	0.6 ± 0.01	0.5 ± 0.02	2	0.5 ± 0.06	0.4 ± 0.04	83	80
Mutant Nod3 ("super-nodular")
2	0.8 ± 0.06	0.6 ± 0.04
3	0.6 ± 0.01	0.6 ± 0.02	1	0.6 ± 0.01	0.3 ± 0.02	100	50
4	0.6 ± 0.01	0.7 ± 0.04	2	0.5 ± 0.05	0.4 ± 0.05	83	57

Authors:

Vasilieva Galina Gennad’yevna

Glyanko Anatoly Konstantinovich

Mironova Nina Vasil’yevna

Putilina Tat’yana Egorovna

Author’s address for correspondence:

Vasilieva Galina Gennad’yevna

Siberian Institute of Plant Physiology and Biochemistry SD RAS

664033, Irkutsk, POBox 1243

Telephone: (395-2)-42-82-56

Fax: (395-2)-51-07-54

E-mail: ustaft@sifibr.irk.ru

Technical College - Bourgas,

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