| Academic Open Internet Journal |
Volume 12, 2004 |
Role of endogenous Phenolic compounds under infection
of pea roots by Rhizobium leguminosarum bv. viceae
Lyudmila E. Makarova, Galina P.
Akimova, Marina G. Sokolova, Galina B. Luzova, Elena G. Rudikovskaya, Vadim
N. Nurminsky
Siberian
Institute of Plant Physiology and Biochemistry, SD RAS
POB 1243,
664033 Irkutsk, Russia, e-mail- ustaft@sifibr.irk.ru
Abstract:
From 5mm sectors of etiolated pea seedlings roots cut along there were
extracted “insoluble” and lipophilic “soluble” phenolic compounds. 48-hours old seedlings subjected to 24 hours
infecting by Rhizobium leguminosarum
bv. viceae were used as the initial
material for the test (non-infected seedlings were used as control). The study
was focused on the “soluble” phenolic compounds impact on Rhizobium propagation in liquid minimal medium. High sensibility of
individual pea roots sectors to nodule bacteria infection was shown (by microscopical
and microbiological methods) to be connected with low values in the tissues and
low activity of the “soluble” phenolic compounds studied. A suggestion was made
that increased concentration of phenolic compounds in question or their high
activity, as well as the increase in the content of “insoluble” phenolic compounds
in root tissues constrain Rhizobium
adhesion, penetration and propagation in relevant root sectors.
Key words: pea; root; Rhizobium leguminosarum; inoculation;
phenolic compound
A particular role in infecting a
legume by Rhizobium bacteria is
attributed to flavonoids synthesized in the host plant root cells [1].
The role of phenolic compounds in microsymbiont propagation remains obscure at all the stages of
symbionts interaction: in rhizoplane and in the roots (inside the infection
threads and after rhizobia removal in the cells of bacteroidal tissue of
forming nodules).
The mechanism
of phenolic compounds impact on bacteria propagation is not clear. It was
assumed that bacteria propagation was not nodD-independent
[2]. It is proven that phenolic
compounds impact on bacteria propagation depends on their structure [2].
Results of the
work [3,4] establish locality of bacteria penetration into the roots, the locus
being conditioned by the presence of symbiont infection sensitive sectors in
the roots. An important role in initiating interaction with microsymbiont is
attributed to flavonoids excreted by the roots to the rhizosphere directly from
the cells of the bacteria penetration zone [4] .
Identified by
Radmoud et al. [4] differences in the
composition of flavonoids secreted by different root zones and producing
different effect on nod-genes expression are likely to be due the differences
in the composition of endogenous phenolic compounds of relevant root sectors.
Possible
phenolic compounds participation in the regulation of Rhizobium penetration in a certain root sector and following
bacteria propagation will depend on the presence within phenolic compounds of
components affecting the above processes. This holds primarily for “soluble”
phenolic compounds. “Insoluble” phenolic compounds, most of which are within
the composition of cell walls [5], are likely to preclude infecting like lignin
[6,7].
Based on the
above our research was aimed at the investigation of root sensitivity to Rhizobium and the impact of “soluble” phenolic compounds
extracted from different root sectors prior to inoculation and at the initial
stage of infecting, as well as determination of the content of “insoluble”
phenolic compounds forming links with cell structures.
This is in
accordance with the goal of the present study – investigation of phenolic
compounds role in predicting a root zone in a plant for Rhizobium bacteria infection.
Material and Methods
For the work
there were used etiolated pea seedlings (Pisum
sativum L.), variety Marat, grown at 22 îÑ on filter
paper moistened with boiled tap water. 2 – day - old seedlings were used as an
initial plant material. The roots were inoculated by Rhizobium leguminosarum bv.
viceae bacteria, strain 245à (obtained from
RSRI of leguminous plants, Orel) by plant irrigation in the cuvettes by water
washing of bacteria cultivar from the solid medium, concentration 2´107 cell/mL, with the estimate 1 mL per
root. Noninfected seedlings of the same age were used as control for inoculated
plants.
Successively cut 5 mm long roots
sectors were taken for the study. The last sector comprised a root part and a
hypocotyl.
In order to identify root zones sensitive to Rhizobium bacteria there was used a
method described in [8] in our modification. After 24 hours of inoculation the
roots were washed by a jet of sterile water, separated from caryopses and, in
order to remove the bacteria lightly associated with root surface, were
thoroughly washed with the phosphate buffer
(pH 7.4) within 15 minutes from the bacteria adsorbed on their surface,
then washed 3-5 times with sterile water.
Absence of Rhizobium colonies
grown on agar medium in the last samples of washing waters was regarded as a
criterion of washing purity. Then
the roots of equal length were cut into 5 mm long sectors and homogenized with
sterile water. Bacteria from the suspense acquired were inoculated on solid
agar medium, 0.05 ml per Petri dish.
Dilution was 1:1000. Intensity of bacteria binding (and perhaps penetration
inside the root) was determined by the quality of grown colonies. One colony
conventionally corresponded to one bacterium adsorbed (or penetrated), which
formed the colony.
Microscopical
analysis was conducted every 24 hours starting from the first day after
inoculation. For this purpose 10-15 plants of equal root length were selected
(based on average parameters for the plants at the development stage under
investigation), separated roots were cut into 5-mm fragments for further
preparation of sections. For the analysis of root hairs deformation both
sections and whole roots fragments equally subjected to coloration were used.
The sections
were obtained manually with the razor and transferred to the object plate with
a water solution drop containing 1% vital blue cresyl dye [9,10]. The sections
were incubated in the dye solution for 2-3 minutes. The dye solution was then
removed with filter paper, the sections were twice washed with distilled water,
and after glycerin application covered with cover glass. The sections preserved the coloring for 1-2
days (at the temperature of 8-10 îÑ).
Sections and
colored root parts were observed through the light microscope («Peraval
interphako”, Carl Zeiss Jena, DDR) and photographed with the photofilm (Fujicolor
Superia X-TRA, 400, Netherlands) with photoextension MFN-11 (Russia) or the
images were stored on a computer hard disk with the video-camera KTP-67
(Russia) and frame fixer on the basis of interface KAMAK.
Phenolic compounds were extracted from the roots of two and three days
old seedlings. The roots were cut into fragments with the interval of 5 mm
starting from the root tip, and combined into individual samples comprising the
sectors, located at the same distance from the root tip. The number of cuttings
in one sample dedicated for phenolic compounds extraction amounted to 250 - 400
with their total fresh weight 1.0 – 2.0 g.
From the roots fixed by 96% ethanol
phenolic compounds were extracted by boiling
80% ethanol, the latter was steamed under the vacuum, the remainder of
HCl was acidified to pH 3-4 and phenolic compounds were extracted by ethyl
acetate. After evaporation ethyl acetate under a cool airstream, the dry
remainder was redissolved in a small amount of 96% ethanol.
Phenolic compounds forming links with the components of cell structures
were liberated as described in [11] from ethanol-insoluble remainder in two
stages, using hydrolysis in 2.0 M solutions of HCl and NaOH. Hydrolysates were
combined, pH was brought to ðÍ 3 – 4 and
phenolic compounds were extracted by the above described method.
The amount of phenolic compounds in stock solution was determined by spectrophotometry
using Folin-Denis reagent [12]. Phenolic compounds content was expressed in mg of root fresh weight. The
calibration curve was plotted using kaempferol (Sigma, USA).
The bacteria were grown first on the solid, then on the liquid medium
according to the earlier method [13]. Components required for Rhizobium propagation were entered into
solid and liquid medium in compliance with the recommendations [14,2].
The impact on Rhizobium
propagation of phenolic compounds contained in the extracts was assessed by the
change of optical density of the bacteria suspense (À 590). To estimate
phenolic compounds impact on bacteria propagation there were used À590 indices acquired 24 hours after the
moment of introducing in the medium of the bacteria for the period of the
exponential phase of their propagation. Measurements were conducted against the
same media without bacteria containing phenolic compounds (for the experiment)
and without phenolic compounds (for control). Optimal for comparison phenolic
compounds concentrations in the media amounted to 2 mg/ml of the medium [13].
The amount of the extract containing the specified phenolic compounds
quantity was calculated based on the parameters of optical density for a unit
of extracts volumes after the reaction with Folin-Denis reagent and calibration
curve obtained for kaempferol (see above).
The parameter À590 = 0.01
corresponded to the number of bacteria placed in the flask prior to each test.
Results and Discussion
The ability to
form nodules on the legumes roots in the course of interaction with ñ Rhizobium is constrained
by certain phases of roots development and the short cells life cycle – targets
sensitive for bacteria [3]. Legumes are capable of rather rigid nodulation
autoregulation controlling bacteria penetration and a number of symbiotic
nodules [15]. Nevertheless, precise
localization of roots zones most sensitive to infection is still being discussed.
Permanent
presence in the medium of seedlings growth of
Rhizobium bacteria contributes
to their continuous interaction with the roots. Therefore, a newly appearing
root sector with higher sensitivity will favor additional bacteria penetration
into the root.
In this
connection we made an attempt to determine the degree of bacteria penetration
into different root sectors along the whole length 24 hours after pea seedlings
inoculation with Rhizobium leguminosarum.
The results of
microbiological analysis display 2 sectors where bacteria associate with root
cells (Fig. 1). They are located at the
distance of 5-10 and 30-40 mm from the
root tip.
Microscopic
studies have shown that mucous bacteria colonies form 24 hours after
inoculation at the distance of 3-7 mm away from the root tip (Fig. 2). Their dimensions
get smaller in the area of growing root hairs.
The root hairs at the beginning of their growth appear approximately at
the distance of 4 mm from the root tip (Fig. 2). A small deformation of the root hairs, which have not finished their
growth yet, was observed at the distance of 8-12 mm from the root tip. It was
best pronounced in the sector located at the distance of 25-40 mm, where root
hairs reached larger dimensions. Bacteria penetration inside root hairs is
likely to begin in this particular sector. Inside these root hairs there were
found small ribbon-like structures, which acquire weak bluish-greenish coloring
with the dye we used. The coloring obtained is considered to be characteristic
of flavonoids [9]. Unfortunately, due
to low microscope resolution, the microscopical method used does not allow us
to confirm the presence of infection threads in root hairs.
In the course of microscopical analyses we also observed that the root
hairs surface adsorbed vital blue cresyl dye. Adsorption varied in the root
hairs differing in age and dimensions. The growing root hairs demonstrated it
primarily in the apical part (Fig. 3), the hairs having reached final
dimensions lacked well pronounced dye adsorption. The phenomenon described may
be a good index of the surface adsorbing activity of root hairs not only in
respect of the dye, but in respect of the rhizobia too, especially so with the
zone of increased adsorption properties of root hairs including sectors with
growing root hairs. This to a certain extent agrees with the literary data on
the response of differing in age root hairs to Nod-factors impact [16]: change
of cytoplasm ðÍ and emergence
of electric potential in infection-sensitive sectors. This reaction is absent in the zone of mature hairs.
As mentioned
above, 24 hours after inoculation pea roots have 2 sectors, on the surface of
which bacteria remain after phosphate buffer treatment (ðÍ 7.4). Solid association of rhizobia and root hairs is
likely to take place in these sectors.
Presumably, the
character of association in the sectors with growing (sector of 5-10 mm) and mature (sector of 30-40 mm)
root hairs may vary. Judging by the results of microscopical observations (see
above), adhesion of rhizobia on their surface is possible. In the last sector,
where no dye adsorption on the root hairs surface was revealed, but they were
found to be substantially deformed and to incorporate small ribbon-like inclusions,
bacteria are likely to penetrate inside root hairs and, apparently, to propagate,
which results in the increase of bacteria quantity (Fig. 1).
The results
acquired allow to consider the sector 5 – 10 mm from the root tip as the most
infection-sensitive for 24 hours upon inoculation.
We have earlier
shown that phenolic compounds extracted from pea roots affect Rhizobium propagation [13]. This
phenolic compounds property proved their biological activity, which is
important for pea roots infection by the given bacteria. The group of “soluble”
phenolic compounds extracted by ethyl acetate turned out to be efficient. The
degree of their impact depended on the age of the seedlings from which phenolic
compounds were extracted and on the concentration of the latter in the medium.
Earlier
acquired data and the data obtained in the course of this work confirm high
concentration of biologically active phenolic compounds group under study in
the root tissues. It significantly exceeds the threshold concentration required
for positive effect on bacteria propagation in the medium investigated [13].
Phenolic compounds, highly active in respect of bacteria were shown to inhibit
their growth in propagation at lower concentrations that phenolic compounds of
lower activity, as the threshold concentration of the former is lower than that
of the latter.
Consequently,
in case of direct impact on the bacteria in the concentrates established in
root tissues phenolic compounds should suppress bacteria propagation.
Bacteria
propagation at the initial stage of their penetration in the root tissues takes
place in the infection threads [17], apparently protecting them from the
influence of excessive phenolic compounds concentrations. Consequently,
phenolic compounds will directly affect bacteria propagation in case of
penetration into the infection threads. However, there are no literary data to
confirm this supposition.
Direct phenolic
compounds impact on Rhizobium at the
initial period of the infection is only possible in case of disturbances in the
formation of infection threads and bacteria penetration from the latter into
the root cells. Perhaps, high phenolic compounds concentrations in the roots
are one of the ways for the plant to control the process of root tissues
infecting by nodule bacteria, preventing their propagation immediately in the
root cells.
The data in
Fig. 4 demonstrate the change in the content and biological activity of the
phenolic compounds group under discussion along the length of the root,
depending on whether the seedlings are inoculated or non-inoculated.
Having compared
the data on root growth with the earlier published results [18], we emphasize
that the highest revealed phenolic compounds activity values in non-infected
roots are observed in the zone with the completely grown cells that started active
differentiation (4-th sector), in inoculated roots – in the zones with the
cells completing extension (3-d sector).
Inoculation is
likely to influence the reduction of phenolic compounds activity in apical
sector of inoculated roots (Fig. 4), though their content in the given sector remains
as high as in the initial roots.
The second
sector of the roots is of interest. Phenolic compounds activity here in the
roots of all the three variants of growing is similar and pretty low (Fig. 4).
In non-infected roots this sector appeared after the first wave of infecting
and, as follows from the data drawn in Fig 1, 24 hours after inoculation is was
also subjected to infection (Fig. 1).
The content of biologically active phenolic compounds there is markedly
lower than in the same root sectors of two other variants (Fig. 4).
It is worth
noting that in the sectors subjected to infection by the former (now they are
sectors 6 and 7, Fig.4), activity and content parameters are close to the same parameters of the second sector. This allows to presume that infection sensitivity of root cells in its
individual sectors, where penetration and propagation of bacteria takes, is
conditioned by low content and activity of the
“soluble” phenolic compounds group under study.
In sectors 3 – 5,
bacteria penetration is apparently unlikely (Fig. 1). Thus, we associate the
role of “soluble” phenolic compounds in hindering bacteria penetration with
their extremely high activity (Fig. 4). The threshold concentration (exceeding
which phenolic compounds are likely to negatively influence bacteria, see
above) should apparently be pretty low. Considerable restriction of sectors 4
and 5 is likely to be conditioned by high concentration of phenolic compounds
discussed (Fig. 1, 4).
It may well be
that the activity observed in respect of the bacteria reproduced is one of the
indices of biological activity of the given phenolic compounds group, which is
of universal importance not only for symbiosis but for the other no less
important processes. High level of phenolic compounds biological activity is
likely to be connected with active metabolism in the cells. This holds for
meristematic and extending cells of the 1-st sector of the roots of all growth
variants, as well as to the cells of the middle part of the roots of 3-days old
seedlings. Inoculation brings about the shift in distribution of highly active
phenolic compounds: from the zone of active cell differentiation to the zone of
the cells being primarily at the stage of growth completion by extension (Fig.
4).
This assumption
can be made based on the results of the work [4], which showed that in the
interaction with bacteria the key role in rhizobia nod- genes expression belongs
to flavonoids excreted by the cells of the root sector where root hairs start
to form. Sector 3 may be such a zone in the roots of non-inoculated pea
seedlings [18]. Based on the above, we can suggest that partially physiological
significance of highly efficient phenolic compounds of the 3-d sector of
inoculated toots consists in their impact on rhizobia nod- genes. This refers to the phenolic compounds that can exit
from the given sector to the rhizosphere within exudates.
Fig. 5 presents
the data on the content of another important phenolic compounds group – that of
“insoluble” phenolic compounds, associated with cell structures.
Incorporation of
non-lignin phenolic compounds in the cell walls of young meristematic and
extending cells was observed when
unfavorable environmental factors affected the plants [11,19,20] and presumably
accounted for the inhibition of root growth in these conditions.
The results of our investigations speak in
favor of participation of the given phenolic compounds group to the limited
infecting of the 1-st sector. This conclusion is based on the association of
the highest (as compared to other variants) parameter of “insoluble” phenolic
compounds content in the given sector with very low bacteria adhesion (Fig. 1,
5). In all the other sectors of
inoculated (except for the 8-th including hypocotyls) the content of “insoluble”
phenolic compounds is comparatively low.
This allows to suppose that the phenolic compounds group discussed in
apical sector performs a protective function, similar to that performed by
another phenolic compounds group - lignin [6,7].
Thus, over the 24
hours of observations (after inoculation) in the pea seedlings roots there are
identified 2 zones with the highest sensitivity to rhizobial infection. Low
values of concentrations and efficiency of the studied group of “soluble”
phenolic compounds in these zones are evidently optimal for rhizobia
penetration and propagation. Their increased concentrations or very high
activity, as well as the increase of the number of phenolic compounds
associated with cell structures are likely to restrict penetration of the given
bacteria and their propagation in the relevant root sectors.
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Figure 2. Bacteria
colonies (marked with arrow) on the root surface at 4-5 mm distance from the
root tip. Right side – root hairs initiating growth. The photograph is taken with
a light microscope. X 175 Coloring by cresyl blue.
Figure 3. Dye (cresyl
blue) adsorption on the surface of growing root hairs (marked with black arrow)
in the root sector at 7.5-9.0 mm from the root tip.
White arrow identifies bacteria
colonyThe photograph is taken with a light microscope X 350.
Figure 4. Content of “soluble” endogenous phenolic compounds (PC)
in noninfected and infected by Rhizoboim
leguminosarum pea root sectors and
their efficiency in respect of rhizobia propagation. Solid line denotes the data on PC
content, dotted line – their biological activity. Values are means ±ses from at least three independent
experiments (n=5 on PC contents, and n=3 on PC biological activity).
Figure 5.
Content of “insoluble” phenolic compounds
in pea root sectors grown by different variants.
“noninf”, “inf” – respectively,
noninfected roots and infected by Rhizobium
leguminosarum (after 24 inoculation).
Values ± means and ses from at least
three independent experiments (n=5).
Technical College - Bourgas,