To determine the content of endogenous IAA the plant material (5 g) was fixed in liquid nitrogen. 3-times extraction by 80% ethanol was performed. The combined extract was softened dry by steam on rotor vaporizer, the dry remainder was washed off by 96% ethanol (3-4 ml) and used to determine IAA by highly efficient method of liquid chromatography /5/.

The experiments were conducted with three- three- six repetitions. The results acquired were processed statistically. The tables and drawings present average arithmetic values and standard deviations.

Results and Discussion

Table 1 presents the results of determination of peroxidase (PO) activity in pee roots 24 h after Rhizobium leguminosarum infection.

Enzyme activity in inoculated plants has been shown (Table 1) to increase both per one root and per gram of raw substance and mg of protein.

The mass of raw root substance compared to control increases in all the pea varieties studied (Tables 2, 3).

Pathogen and host interaction is known to cause a considerable increase in the activity of PO which play a key role in the plant protective mechanisms /6/. Nevertheless, Rhizobium invasion in the legume roots takes place in the course of a highly harmonious infection process that does not provoke the protection reaction of the host and bacteria pass along the infection thread towards the “target cell” /7/. Moreover, legume root has an area of the highest sensitivity to infection /8,9/.

With this in view we made an attempt to determine the degree of rhizobia penetration in various roots sectors along its length 3, 6 and 24 h after inoculation of pea seedlings by Rhizobium leguminosarum.

As shown in fig. 1, maximal penetration of rhizobia is observed in the area 5-15 mm away from the root tip 3 and 6 hours after inoculation. After 24 h there are observed two peaks in the number of the bacteria penetrating in the root. The first one is apparently conditioned by new bacteria penetration and the second one – shifted in the area of 20-35 mm from the root tip as a result of its growth – is evidently a number of bacteria that penetrated after 3 and 6 (and, probably, more) hours after inoculation and reproducing inside the root during this period.

The results acquired allow to consider the area 0 -20 mm from the root tip most sensitive to infection and thus, further investigation would be more efficient with this root characteristics taken into account. Table 4 illustrates PO activity in the root sectors differing in Rh. Leguminosarum infection sensitivity.

Enzyme activity is shown to have different orientation of changes. In the sectors 0-20 mm from the root tip PO activity in inoculated pea plants of Rondo, Torsdag and Marat varieties slightly reduces, and in more differentiated sectors (20-40 mm from the root tip) significantly grows, which, evidently, affects the general enzyme activity determined in the whole roots.

Thus, PO activity reduction in the root area most sensitive to infection does not only correspond to maximal Rhizobium penetration in this sector, but is very likely to contribute to it.

Such different orientation of PO activity changes in pea roots sectors in the course of interaction with Rhizobium necessitates the comparison of these results with the enzyme response in pea plants under bacteriosis.

PO activity in the sectors of such roots was shown to significantly increase as compared to the plants whose seeds were subjected to additional sterilization prior to sprouting (Table 5).

The increase of enzyme activity along the whole length of the root has an intense tendency towards augmentation as the plants grow as far as 5 days. The results obtained prove that peroxidase complex in the given interaction acts as an indicator of plant stress in response to biotic factor /6/.

Therefore, it is logical to assume that peroxidase activity reduction in the root sector most sensitive to Rhizobium, infection in inoculated plants apparently assists in bacterium penetration in the root. Peroxidase generalized in the root sector non-sensitive to infection is likely to have a direct anti-bacterial effect /1/, as is the case with the roots infected by bacteriosis – characterized as a rule by oxidation explosion, active oxygen forms formation, lignification of cell walls. This obviously prevents systemic distribution of Rhizobium infection in the root and protects the plant from excessive infection.

Hence, the results prove that the orientation of peroxidase activity changes differs in pea roots zones differing in sensitivity to Rhizobium leguminosarum infection. At the same time enzyme activity decrease in the sector closer to root tip is rather specific and unlike activity increase in the course of interaction of such incompatible partners as plant-parasite it may not be regarded as a certain general response to stress.

In case of symbiotic relations PO is likely to condition the mechanism of nodulation self-regulation controlling its degree via Rhizobium bacteria penetration in the sensitive root sector and preventing it in other root zones.

The level of peroxidase activity may be controlled by IAA, which changes enzyme specificity from peroxidase to oxidase converting peroxidase in highly specific oxigenase, which generates free radicals that are necessary for the development of the plants /10/ – the process apparently taking place in pea seedlings roots under Rhizobium Infection. At that orientation of peroxidase reactions depends on the distance from the root tip and sensitivity of this sectors to rhizobial infection. It is important to note that it is in Rhizobium infection sensitive root sectors that IAA content increases (table 6) with IAA prolonging cells expansion inoculated plants /9,11/, whereas in the root sector non-sensitive to infection IAA content increase is not observed. IAA acting as a trigger in oxidation reaction catalyzed by PO /10/ along with the former plays a significant role in the mechanisms of root nodulation self-regulation.

Therefore, the data acquired allow to conclude that one of the self-regulations mechanisms of pea roots nodulation under infection by Rhizobium may be set in the regulation of the activity of peroxidase, which acts selectively and conditions the mechanism of nodulation self-regulation controlling the number of nodules in the root. Further study of functional peculiarities of PO, and particularly its role in the oxidation of IAA and phenolic compounds, at that not only citoplasmatic, but membrane-associated as well, will contribute to understanding of finer mechanisms of symbionts' interaction and, probably, will allow to characterize it as a marker enzyme to determine the area of root sensitivity, and partners’ compatibility for efficient symbiosis.

1. Averyanov А.А., Lapikova V.P. // Oxygen radicals in chemistry, biology and medicine. Riga, Latvia. 1988, pp. 203-222.
2. Buffard D., Estault R., Kondorosi A. // World Journal of Microbiology and Biotechnology. 1996, v.12, pp. 175-188.
3. Lebedeva O.V., Ugarova N.N. and Berezin I.V. // Biochemistry. 1977, v.42, pp. 1372-1379 (in Russian).
4. Ho L-C, Wang J.L, Shendler M. and Loh J.T. // Plant J. 1994, v. 5, pp. 873-884.
5. Procedures on Phytohormones Identification. 1988. Kiev, AS Ukr. SSR, Ukraina, 78 p.
6. Savich I.M. // Peroxidases – plant stress proteins. 1989, v. 107, pp. 406-417 (in Russian).
7. Verma D.P.S. // Plant Cell. 1992, v.4, pp. 373-382.
8. Bhuvanaswari T.V., Turgeon B.G., Bauer W.D. // Plant Physiology. 1980, v. 66, pp. 1027-1031.
9. Sokolova M.G. // Physiological peculiarities of initial stages of pea roots infection by Rhizobium leguminosarum at various temperatures. 2001, рр.11-13. Abstract of Ph.D. Biology thesis. Irkutsk, Russia (in Russian).
10. Rogozhin V.V. // Physiological-biochemical mechanisms of formation of hyobiotic states in higher plantsю. 2000, рр. 49-50. Abstract of Dc.S. Biology thesis, Irkutsk, Russia (in Russian).
11. Akimova G.P., Sokolova M.G., Nechaeva L.V. // Plant Physiology. 1999, v.46, pp. 806-810 (in Russian).