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Volume 14, 2005

 

 

PRODUCTION OF SUPEROXIDE RADICALS IN PEA SEEDLINGS 

 (Pisum sativum L.)  AT INOCULATION WITH NITROGEN - FIXING BACTERIA STRAINS DIFFERING IN COMPATIBILITY

 

Galina G. VASILIEVA, Anatoly K. GLYANKO, Nina V. MIRONOVA

 

Siberian  Institute of Plant Physiology and Biochemistry SD RAS

 

POB 1243, Irkutsk, Russia, 664033,  e-mail - ustaft@sifibr.irk.ru

 

Abstract:

 

The production of superoxide radicals (O2-.) in  pea seedlings (Pisum sativum L.) at inoculation with compatibility differing strains of nitrogen-fixing bacteria (Rhizobium leguminosarum) was studied. The study revealed the ability of O2-.  to be generated by uninoculate seedlings. At inoculation with compatibility- and effectiveness-differing strains of nitrogen-fixing bacteria differences were observed in the intensification of the process. The question of the involvement of the O2-. production process in early formative stages of symbiotic relations is discussed.

 

Key Words: Superoxide radical; inoculation; activity reducing cytochrome c

 

There has been recent evidence that in early formative stages of symbiotic relations with nitrogen-fixing bacteria a leguminous plants can affect the infection process accomplished by the microsymbiont by triggering a deffense mechanism similar to the "hypersensitive  reaction" (HSR) observed  during the interaction of incompatible organisms: plant-pathogene [1].These authors have shown that subsequent to the nodulation onset in some cortical cells of the root there is an increase of the number of aborted infection threads, and both symbionts undergo necrosis simultaneously. Necrotic cells accumulate phenol compounds and proteins associated with defense mechanisms in plants. It is anticipated that the triggering of the HSR forms part of the mechanism by which the plant controls the infection process and hence the nodulation.

    In many publications it has been demonstrated that the HSR is closely related to an enhancement of the generation of active oxygen species (AOS), the most important of which is O2-. .  However, this process is now better understood in the plant-pathogene system [2,3,4,5,6]. Enhanced activation of oxygen in response to infection is observed in different plant species and in infection processes of a different nature (fungal, bacterial and virus diseases, and affections by nematodes). However, the question of the possible role of protective systems in self-regulation of the infection and nodulation processes in symbiotrophic organisms is poorly understood.  In our view, this issue is of important significance for the understanding of  mechanisms for symbiotic relations. According to Brewin  et al. [7] the problem of the penetration of rhizobia into tissues may be formulated as the problem of eliminating induction of  protective systems of the host plant accompanied by the generation of free radicals of oxygen. Therefore, the objective of this paper is to investigate the production of O2-.  by pea seedlings  as a likely factor of the primary response of the plant cell to the effect of nitrogen-fixing bacteria strains with different compatibility and effectiveness.

 

Materials  and  Methods

 

   Seedlings of pea (var. Marat) were grown in dishes on wet filter paper in the thermostat at the temperature of 22o C  during two days after inoculation. Inoculation of the roots of 2-day old seedlings was done with nitrogen-fixing bacteria Rhizobium leguminosarum bv. viceae, with an effective (strain 250a) and ineffective (strain 249b) pea-specific strains. In addition, inoculation was accomplished with pea-incompatible strains:

1. Rhizobium leguminosarum bv. viceae, a wild strain specific for bean (Vicia faba L.);

2. Rhizobium leguminosarum bv. phaseoli, a wild strain specific for kidney bean (Phaseolus vulgaris L.).   

   Dilution of the culture was used in inoculation: 2 x 108 cells/ml. The volume of bacterial suspension in all variants was 1 ml of suspension per root. Effective and ineffective pea- specific strains Rhizobium leguminosarum bv. viciae  and strain 348a (Rhizobium leguminosarum bv. trifolii) were received from the collection of the All-Russian Institute of Agrocultural Microbiology (St. Petersburg, Russia), while bean and kidney bean- specific wild strains were local (environs of city Irkutsk, Russia).

   The determination of O2-. is based on its cytochrome c reducing activity [8]. Therefore, for evaluating the generation  of O2-.  us the root and epicotils, reduction activity cytochrome c (cyt. c RA)  was measured  1 and  2 days after inoculation. To determine cyt. c RA, 10 seedlings of each variant  were flushed with running water and then with distilled water, and were slightly drieed using filter paper. Detached roots (25-68 mm length) and epicotyls (5-25 mm length) were cut into segments  (1.0-1.5 mm thick) and were placed into microdishes of volume 6 ml with the incubation medium of the following composition: 10 mM K-phosphate buffer, pH 7.4; 50 mM NaCI; 10 mM NaN3; 0.1 mM EDTA, and 10 μM cyt. c ("Aldrich", USA). The  cytochrome c reducing activity was recorded  with the "Specord" UV VIS spectrometer ("Carl Zeiss", Germany) in microdishes 1 = 2.0 cm. Calculations were performed by using the difference coefficient of extinction between the oxidized and reduced forms of cytochrome c with E550 = 2.21x104 M-1 cm-1.

    In this paper, arithmetical means values based on data from 3-6 assys are presented (M±SD).

 

Results

 

    Based on the assays made, we detected the ability of roots and epicotyls of uninoculate  pea seedlings to reduce exogenous cytochrome c  (Table 1).The largest cyt.c manifests itself during the first 24 hours of germination of seeds and decreases with the age of seedlings. Also, cyt.c RA was invariably by factor of 2-3 higher in epicotyls than roots.

    For evaluating  the dependence of cyt.c RA of seedlings on the exposure time in the cytochrome c solution, determinations were made during 1 hour, as well as 2, 3, 4, 6,  18 and 24 hours of exposure. It was established that the the reducing cytochrome c activity occured mainly during the 1-st hour of exposure (Table 1). Subsequently, comparisons of the various variant for cyt. c RA were therefore made 1 hour after the in the cyt. c solution.

   To ascertain how inoculation with effectiveness- and compatibility-differing strains of  nitrogen-fixing bacteria affected the production of O2-. in  pea seedlings, the reduction of cytochrome c for each variant was measured 24 and 48 hours after inoculation. It was found  that the convenient observing time of the production of O2-. all variants constituted 24 hours elapsed after inoculation (Table 2).

   Inoculation with effective strain 250a accompanied by a slight increase in cyt.c RA: by 20% and 36%, respectively, for the root and epicotyl (Tab.2). Inoculation with ineffective strains 249b led to a substantial increase in cyt.c RA: by 97% and 129%, respectively, for the root and epicotyl. A more complicated picture was observed in the case of inoculation with pea-non-specific strains. Thus inoculation of seedlings with bean-specific wild strain led to a slight increase in cyt.c RA (by 18%) and to an increase by 52% in epicotyls. Rhizobium leguninosarum bv. phaseoli inoculation caused increasing cyt.c RA in both a organs of seedlings:  by  2.5 times in roots and by 2.8 times in epicotyls (Table 2).

 

Discussion

 

  The ability of uninoculate seedlings of pea to restore cyt.c (Table 1) point up the production of O2-. as a physiological norm because AOS are known to be the necessary link of the metabolic chain in the plant organism, and their level influences directly or indirectly such fundamental processes as the growth and development of the organism, and the maintenance of AOS on the required and safe level for the cell is a vital strategy of plant [9, 10]. Previosly the production of O2-. in the root of an intact  pea seedlings was detected and measured using the oxidation of adrenalin inhibited by superoxide dismutase (SOD) [10].

  From the microbiological characteristic of nitrogen-fixing bacteria strains it is known  that strain 250a that is effective in its nitrogen-fixative ability, is also a medium-virulent and medium-competitive one. Strain 249b that as ineffective in its nitrogen-fixing ability, shows a high virulence and competitivenes. We believe that the differences in activation of the O2-. generation process in pea seedlings, when they are inoculated with these strains, may be attributed to their different virulence. Thus, production of  AOS is observed at the earliest stages of symbiotic relations. 

   At this juncture in our research, it is difficult for us to explain the differences in the O2-. production in  pea seedlings when they are inoculated by incompatible strains of nitrogen-fixing bacteria, because the question as to whether some of the incompatible strains, which we have investigated, possess virulence with respect to pea roots, or they make only surface contact with the host plant on the level of attachment, for example, is obscure. An abrupt increase in radical production in the plant organism before or during the penetration of pathogen microorganisms into it has now been demonstrated on several objects [2]. However, this question remains open for bean plants in the case of  their inoculation with incompatible strains of nitrogen-fixing bacteria;  therefore, microbiological studies are needed in this case.

   Noteworthy is the fact that inoculation causes a change in cyt.c RA not only in roots but  also in epicotyls, the organs which are not directly associated with the infection process, subsequent nodulation, and nitrogen-fixation. An enhancement of the O2-.  production in epicotyls is suggestive of some intracellular signal mechanism that operates during the interaction of the root with root nodule bacteria. It  can be connected with   protective response, witch  in including to prevent systemic  spread of rhizobia (induced systemic resistance) [11,12].

 

                                     

                                  Literature cited

 

1. Vasse J., De Billy F.,  Truchet G. // The Plant Journal, 1993, v. 4,  pp. 555-566.

 

2. Aver,yanov A.A. // Recent Advances in Biol., 1991, v. 111, pp. 722-737 (in Russia).

 

3. Lapikova V.R. , Aver,yanov A.A. // Res. Papers  Acad.  Sci., 1993, v. 329, pp. 253-255 (in Russia).

 

4. Mehdy  M.C. // Plant Physiol., 1994, v. 105, pp. 467-472.

 

5. Bottin A., Veronesi C., Pontier D., Esquerre-Tugaye M-T., Blein J-P., Rusterucci C.,  Ricci P. // Plant Physiol.  Biochem., 1994, v. 32, pp. 373-378.

 

6. Glumova N.V., Guzhov N.V., Merzlyak M.N., Chivkunova O.V. // Plant Physiol.,1994, v. 41, pp. 130-134  (in Russia).

 

7. Brewin N.J., Bolonos L., Daniya P., Gardner C.D., Hernandez L.E., Kardailsky I.V., Rathbun E.A.,  Sherrier D.J.  In: Nitrogen fixation: fundamentals and applications (eds.: Tikhonovich I.A., Provorov N.A., Romanov V.I., Newton W.E.), 1995, p.455-460. Kluwer Academic Publishers, Dorrdrecht, Boston, London.

 

8. Doke N. // Physiol. Plant Pathol.,1983, v. 23,  pp. 345-357.

 

9. Askochenskaya N.A. , Chetvericov A.G. // Physiol.  Biochem. Culture Plants. 1989, v. 20, pp. 572-577  (in Russia).

 

10. Aver,yanov A.A. // Plant Physiology, 1985, v. 32 , pp. 268-273  (in Russia).

 

11. Lamb  C.J. //  Cell, 1994, v. 76, pp. 419-422.

 

12. Van Loon L.C., Bakker P.A., Pieterse C.M.J. // Annu. Rev.  Phytopathol.,1998, 36, pp. 453-483.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 1. Reduction of cyt. c by pea seedling depending on exposure  time in the cyt. c solution and on the age of  seedlings

 

Age of seedlings,

days

Exposure time in

cyt. c solution, h

Reduced  cyt. c (nmol/g fresh tissue)

Root

Epicotyl

 

2

1

36.5 ± 2.8

-

2-3

54.4 ± 5.1

-

4-6

47.5 ± 4.2

-

18-24

54.3 ± 5.3

-

 

3

1

21.6 ± 2.0

62.1 ± 5.9

2-3

28.8 ± 2.9

72.6 ± 6.9

4-6

22.4 ± 1.9

70.9 ± 7.1

18-24

14.7 ± 1.4

65.9 ± 6.5

 

4

                                         

1

12.3 ± 1.0

28.6 ± 2.4

             2-3                       

        12.2 ± 1.1              

        44.0 ± 4.1

             4-6               

        14.2 ± 1.3                 

        27.6 ± 2.4

 

TABLE 2. Effect of compatibility-differing strains of nitrogen -fixing bacteria on reducing activity cytochrome ñ of  pea seedlings 

 

Time of action

of inoculant,

days

Reducing activity cytochrome c

nmol/g fresh tissue .  hour-1

% of uninoculated

Root

Epicotyl

Root

Epicotyl

Control (without inoculation)

            1

21.6 ± 2.0

62.1 ±  5.9

 

 

            2                   

12.3 ± 1.0

28.6 ± 2.7

 

 

 

Rhizobium leguminosarum bv. viceae, strain 250a (effective)

            1                                              

26.0 ± 1.3

84.6 ± 7.0

120

136

            2                    

9.5 ± 0.2

47.6 ± 4.5

77

166

 

Rhizobium leguminosarum bv. viceae, strain 249b (ineffective)

            1

42.6 ± 4.0

142.3 ± 10.1

197

229

            2                                   

16.9 ± 1.5

49.5 ±  4.1

137

173

 

Rhizobium leguminosarum bv. viceae, bean-specific wild strain

            1                  

25.5 ± 1.2

94.2 ± 8.6

118

152

            2                              

11.4 ± 1.0

54.6 ± 4.9

93

191

 

Rhizobium leguminosarum, bv. phaseoli, wild strain

            1                  

54.5 ± 4.3

171.6 ± 13..3

252

276

            2                                

12.8 ± 0.8

84.3 ± 7.9

104

295

 

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