TEMPERATURE IMPACT ON THE DEFORMATION OF ROOTS HAIRS with Rhizobium - INOCULATED PEA (Pisum sativum L.) SEEDLINGS

 

Lyudmila E. Makarova, Vadim N. Nurminsky

 

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

664033 Irkutsk, Russia,

e-mail – makarova@sifibr.irk.ru

 

ABSTRACT. Using light microscopy methods we monitored the response of root hairs (deformations) in pea (Pisum sativum L.) seedlings  grown at 22 and 8 ˚С, 24 hours after inoculation by the bacteria  Rhizobium leguminosarum bv. viceae. There were noticed differences between the roots grown at different temperatures – they manifested different deformation degree (twists, bends, etc.). It was suggested that inhibition of pea roots infection by nodular bacteria at lower temperature was caused by the slowed down of growth of both the root itself and root hairs, as well as decelerated increase of root hairs zones. 

 

Key words: Rhizobium leguminosarum bv. viceae- inoculation – root hairs - temperature

 

In the course of establishment of symbiosis between legumes and Rhizobium the temperature factor is of great importance, as it affects bacteria propagation in the soil [1, 2] of infection threads formation [3] and nodules formation [3,4].

The way of rhizobia penetration into pea root tissues is assumed to pass through root hairs cells [5]. Deformation and twisting of root hairs act as first morphologically distinguishable changes induced by rhizobia. Deformation was established to emerge only at a certain stage of root hairs development: at the stage of growth termination  [6].

Taking into account temperature impact on the speed of root growth and development [4], one may assume that slow-down of these processes caused by the temperatures, unfavorable for the plants, may influence its infecting with Rhizobium.

The impact of temperature inhibiting formation of symbiotic relations between legumes and Rhizobium may apparently begin with its influence on the growth of root hairs, their deformation and bacteria penetration into them.

With this in view the present work was targeted at the study of optimal (22 ˚С) and lowered (8 ˚С) temperatures on the development of root hairs response to infection with Rhizobium. We monitored this response by the development of deformations of root hairs 24 hours after infecting.

Material and methods

Ethyolited pea (Pisum sativum L.) seedlings, Marat variety, grown in the darkness on filter paper wetted by boiled tap water were used for the investigation.  As initial material there were used seedlings, grown in one case during 2 days at 22 ˚С, and in the other case during 7 days at 8 ˚С (starting from the soaking moment), until their root length reached 2.8-3.0 cm. The response to inoculation by Rhizobium was studied 24 hours after it began. Non-infected plants of the same age were used as control. 

Inoculation was performed by bacteria R. leguminosarum  bv. viceae strain 250а – СIAM 1026 (received from the collection of Agricultural Microbiology VNIISKhM ;’S-Petersburg, Pushkin). The inoculate, containing bacteria R. leguminosarum (titre 2 ´10⁷ cells / ml of medium), was introduced only once at the beginning of the experiment in small amount (1 ml/plant) of liquid – washing residue of bacteria cultivar grown in hard agar substrate by the method [7].

Microscopic analysis was performed 1 day after the inoculation. There were selected plants with root length close to the average value of the development stage under study, the root were separated, for more distinct picture of interaction between bacteria and root cells while determining adhesion and penetration zones there was used dying by the method described in [8,9]. To this end the roots were placed in the water solution containing 1% vital blue cresyl dye  (“Merck”, Germany) on the object plate. Roots incubation in the dying solution lasted for  2-3 min. The paint solution was removed with the help of filter paper. The roots were twice washed by distilled water, then they were coated with glycerin and covered by cover glass. Colored roots, on the surface of which there were found morphological changes of root hairs caused by inoculation, were observed through the light microscope («Peraval interphako», Carl Zeiss, Jena, GDR).

The adhesion zone was determined by the accumulation of nodular bacteria on the root surface, the penetration zone was determined by the degree of root hairs deformation and emergence of infection threads in them. 

Images were obtained with the digital photocamera Nikon Coopix 4300 (Japan), with the use of photo-nozzle MFN-11 (Russia) set into the microscope.  

 

Results and discussion

 

We have earlier noticed negative impact of lowered temperature on the development of symbiotic relations between pea and rhizobia; it inhibited infecting and nodulation processes [10, 4].

The root growth is slowed down at lowered temperature [11]. Deceleration of growth processes under low temperature is apparently related to hindered development of symbiotic relations between plants and rhizobia. Presumably, at low temperature in this case along with deceleration of root growth speed root hairs growth speed also goes down [3], initiation of infection threads formation and their growth also slow down [12]. According to F. Zhang and G. Smith [12], in the process of symbiosis, the infecting stage including rhizobia penetration and infection threads development is one of the most sensitive stages to low temperature impact.

It is currently believed that rhizobia penetration in pea root tissues takes place through the root hairs, and sensitivity of root hairs to rhizobial infection depends on the hairs’ age [6]. Mature root hairs are insensitive. It is suggested that first stages of interaction between symbionts associated with bacteria adhesion on the surface of root cells also happen in the zones, where root hairs have yet begun to grow [13].

A regularity between root length and beginning of formation and degree of development of root hairs was observed [3].

Our plant object – pea – demonstrated formation of the first root hairs when roots length achieved about 2.2 cm. With the root length of about 3 cm there appeared a zone of root hairs, whose maximal size approximated 20 –30 μm.

In the present experiment there was also marked the impact of seedlings growth temperature on the root hairs growth. Besides, there was identified its impact on the root hairs shape at the beginning of their growth and length of the root hairs zone. Thus, at the temperature of 22 ˚С, optimal for the growth, the root hairs of 10-20 μm  in size, were of conical shape, later, with further growth they acquired cylindrical shape with rounded apical tip. At lower temperature (8 ˚С) the root hairs after conical shape largely acquired mushroom-like shape (with the size of about 20-30 μm ) (Fig.1А, B), but in the course of further growth they also became cylindrical in shape. One could observe some curvature of their surface even without interaction with rhizobia (Fig.1А).

The variant with the temperature 22 ˚С manifested better pronounced deformation changes in the root hairs in response to bacteria penetration 24 hours after the inoculation. This is illustrated by Fig. 1B и 2C, with the images of root hairs in the root sections with the best expressed response to inoculation in the seedlings grown at 8 and 22 ˚С.

It was observed that in comparison with low-temperature variant at the temperature 22 ˚С during 25 hours after inoculation the roots together with considerable length increase developed a pretty vast zone of root hairs. With the average root growth of 20.0 ± 2.5 mm along the length it reached approximately 20-25 mm. 

In the variant with the temperature 8 ˚С root growth during the same period amounted on average to 3.0 ± 0.4 mm, and the sector with root hairs reached 5-7 mm in length. Only seedlings with root length of 35-36 мм, which made up only 10% of the total number of the seedlings grown, demonstrated the increase of root hairs zone up to 13.5 ± 1.0 mm.

At the temperature 22 ˚С the first signs of epidermal cells, which initiate root hairs, began to show approximately from 4-5 mm from the root tip. In this part of the root, as well as the part, adjacent from the apex side, one could see bacteria accumulations. However, further from the root tip the number of bacteria located in the intersections of root hairs decreased progressively with the increase of their size.

Visible response to inoculation in the form of deformation first manifested itself at the distance of approximately 8-10 mm from the root tip. Here and further, up to about 20-25 mm from the root tip root hairs were insignificantly deformed. Deformation largely showed in the emergence of small transverse constrictions, twists in apical sector of root hairs (Fig. 2 А, B).

 Further, starting from 22-25 mm, the root hairs achieved fairly large dimensions (up to 200 μm), though there were also formed smaller root hairs (60–90 μm) (Fig. 2 B). In this zone, on average 5 – 9 mm long, root hairs manifested the best pronounced deformations. Besides, on some root hairs which achieved 60-90 μm, one could see little strainings - infection threads, which confirmed that the rhizobia penetration process had started  (Fig. 2 C). It is worth noticing that with the size of about 200 μm  (Fig.2 C), the root hairs subjected to the most intense deformation practically did not contain any inclusions in their upper segment. This may be connected with the intensification of tip growth of the cell coat of the root hairs under the impact of rhizobial Nod –factors and transfer of infection threads to the crust cells [6].

Both at optimal and lowered temperature, the plants had adhesion bacteria on the surface of the root sector, starting from its apex towards the root hairs zone. The first sensitive segment of the roots in these conditions coincides with the section at  5-10 mm from the root tip. On the surface of this sector one could see bacteria accumulations lessening with removing from the root tip. The majority of root hairs initiating growth did not suffer any deformation. These facts may be regarded as a proof of the fact that this sector hosts rhizobia adhesion on the surface of root cells, but not their penetration.

At the temperature 8 ^оС during 24 hours after inoculation the average length of root hairs significantly increased. In the roots of initial seedlings with the root hairs zone length of about 2.0 mm, the length of the latter amounted to 10-20 μm. 24 hours after the inoculation, on a small root sector (5-7 mm long with the root length ranging from  31 to 34 mm), which began at the distance of 9.0 ± 1.0 mm from the root tip, one could observe root hairs of different length: from 10-20 to 60-80 μm. When the root achieved 35-36 mm length, we observed emergence of a small number of root hairs, whose length varied from 90 to 120 μm (Fig.1 B). These root hairs were found only in the narrow root section, about 1.5 ± 0.5 mm long at the approximate distance of 16.0 ± 1.0 mm from the root tip. It should be emphasized that this is where infection threads appeared (Fig.1B).

 It was also revealed that with the same root size, root hairs of inoculated plants were considerably longer. Intensification of root hairs growth with inoculation may apparently be accounted for by the impact of rhizobial Nod –factors [14].

Thus, inoculation was shown to intensify root hairs growth. With the root length increase the length of root hairs zone and dimensions of mature root hairs also enhanced. The temperature affected the speed of root growth, the size of root hairs and their length.  In this connection, the results acquired allow to suppose that hindering of infection process under lowered temperature may be slowing down of root hairs development caused by low rate of root growth. Of importance is also decelerated increase of root hairs zone, which might result in considerable limitation of the number of root hairs, which are sensitive to infection.

 

Literature cited

1.      Mishustin E.N., Shilnikova V.K. Nodular bacteria and process of inoculation. / Moskow, Nauka. 1973. / in Russian/.

2.      Sadowsky M.J., Graham P.H. // The Rhizobiaceae. Molecular Biology of Model Plant-Associated Bacteria / Ed.H.P. Spaink, A. Kondorosi, P.J.J. Hooykaas / Dortrecht/Boston/London, Kluwer Academic Pubishers. 1998, p.

 179-197.

3.      Kumarisinghe RMK, Nutman PS.. // Journal of experimental Botany. 1979. V. 30, p. 503-515.

4.      Makarova L.E. // Agrochemistry. 2001. N 12, p. 24-28.

5.      Hadri A-E., Spaik H.P.,.Bisseling T., Brewin N.J // The Rhizobiaceae. Molecular Biology of Model Plant-Associated Bacteria / Ed.H.P. Spaink, A. Kondorosi, P.J.J. Hooykaas / Dortrecht/Boston/London, Kluwer Academic Pubishers. 1998, p.373-387.

6.      Hadri A-E., Bisseling T. // The Rhizobiaceae. Molecular Biology of Model Plant-Associated Bacteria / Ed.H.P. Spaink, A. Kondorosi, P.J.J. Hooykaas / Dortrecht/Boston/London, Kluwer Academic Pubishers. 1998, p. 435-464.

7.      Berestetskii V.A.// Methods of new Rhizobium leguminosarum strains acquisition and their efficiency evaluation./ Leningrad, VNIISKHM. 1976, p17-23 /in Russian/.

8.      Kartush B. // Protoplasma. 1975, v. 86, p. 371-379.

9.       Scott M.G., Peterson R.L. // Canadian Journal of Botany. 1979, v. 57, p. 1063-1075.

10. Makarova L.E., Luzova G.B., Lomovatskaya L.A. // Russian Journal of Plant physiology. 1998, v. 45, p. 824-832 /in Russia/.

11. Makarova L.E., Latysheva S.E., Ekimova E.G. // Russian Journal of Plant physiology. 2003, v. 50, p. 291-295 /in Russia/.

12. Zhang F., Smith D.L. // Journal of experimental Botany. 1994, v. 45, p. 1467-1473.

13. Bhuvaneswari T.V., Turgeon B.G., Bauer W.D. // Plant Physiolоgy. 1980, v. 66, p. 1027-1031.

14. Kurkdjian A.C. // Plant Physioljgy. 1995. V. 107, p. 783-790.

 

 

 

Figure 1. Root hairs on the pea seedlings roots grown at 8 ˚С.

А – non-infected plants. The image was obtained at the distance of 15-16 mm from the root tip. Root length achieved 36.0 mm.

В - Rhizobium – infected plants. The image was obtained at the distance of 14-15 mcm from the root tip. Root length achieved 36.0 mm.

The arrow designates the infection thread with rhizobia, which passes inside the root hair.

 

 

 

 

 

 

 

 

 

Figure 2. Root hairs in different sections of inoculated pea seedlings roots grown at 22 ˚С. 

The photographs show root hairs in the sections located at 10-15 (А), 15-20 (В), 20-25 (С) mm from the root tip. The root was 45.0 mm long. The arrow designates the infection thread with rhizobia, which passes inside the root hair.

 

 

 

 

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