Plant Physiol. (1999) 119: 565-574
Rhizosphere Bacteria Enhance Selenium Accumulation and
Volatilization by Indian Mustard1
Mark P. de Souza,
Dara Chu,
May Zhao,
Adel M. Zayed,
Steven E. Ruzin,
Denise Schichnes, and
Norman Terry*
Department of Plant and Microbial Biology (M.P.d.S., D.C., M.Z.,
A.M.Z., S.E.R., D.S., N.T.), and College of Natural Resources Center
for Biological Imaging (S.E.R., D.S.), University of California, 111 Koshland Hall, Berkeley, California 94709-3102
 |
ABSTRACT |
Indian mustard (Brassica
juncea L.) accumulates high tissue Se concentrations and
volatilizes Se in relatively nontoxic forms, such as dimethylselenide.
This study showed that the presence of bacteria in the rhizosphere of
Indian mustard was necessary to achieve the best rates of plant Se
accumulation and volatilization of selenate. Experiments with the
antibiotic ampicillin showed that bacteria facilitated 35% of plant Se
volatilization and 70% of plant tissue accumulation. These results
were confirmed by inoculating axenic plants with rhizosphere bacteria.
Compared with axenic controls, plants inoculated with rhizosphere
bacteria had 5-fold higher Se concentrations in roots (the site of
volatilization) and 4-fold higher rates of Se volatilization. Plants
with bacteria contained a heat-labile compound in their root exudate;
when this compound was added to the rhizosphere of axenic plants, Se
accumulation in plant tissues increased. Plants with bacteria had an
increased root surface area compared with axenic plants; the increased
area was unlikely to have caused their increased tissue Se accumulation because they did not accumulate more Se when supplied with selenite or
selenomethionine. Rhizosphere bacteria also possibly increased plant Se
volatilization because they enabled plants to overcome a rate-limiting
step in the Se volatilization pathway, i.e. Se accumulation in plant
tissues.
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INTRODUCTION |
Se pollution is a major concern for agriculture in California and
other parts of the western United States because irrigation of
seleniferous soils derived from shale rocks has led to leaching of Se
and other naturally occurring trace elements such as B and As into the
drainage water (Aubert and Pinta, 1977
; Presser and Ohlendorf, 1987).
The predominant form of Se in agricultural drainage water is selenate
(McNeal and Balisteri, 1989), which is known to bioaccumulate with
significant ecotoxicological effects (Presser and Ohlendorf, 1987). At
the Kesterson, California, reservoir, Se-laden agricultural runoff
accumulated in the food chain, resulting in the deformity and death of
birds and fish (Ohlendorf et al., 1986; Saiki and Lowe, 1987).
Se is also a significant contaminant in industrial wastewater (Manceau
and Gallup, 1997; Hansen et al., 1998). Constructed wetlands have been
shown to efficiently remove Se from contaminated wastewater: 89% of
the Se entering a constructed wetland as selenite-contaminated oil
refinery effluent was removed (Hansen et al., 1998). A large proportion of the Se removed by the wetland was accumulated in plant
tissues and sediments, and it was estimated that 10% to 30% of the
removed Se was volatilized. Se volatilization is the process by which
inorganic Se is converted to volatile forms (Lewis et al., 1966; Evans
et al., 1968; Lewis, 1971; Frankenberger and Karlson, 1994; Terry and
Zayed, 1994). Dimethylselenide, the predominant form of volatile Se, is
500 to 700 times less toxic than inorganic forms of Se (McConnell and
Portman, 1952; Ganther et al., 1966; Wilber, 1980).
Phytoremediation, the use of plants to remove, stabilize, or detoxify
pollutants, is an environmentally responsible and efficient way to
clean up Se-contaminated soil and water (Terry and Zayed, 1998). Many
plant species have been shown to very efficiently accumulate and
volatilize Se (Terry et al., 1992; Pilon-Smits et al., 1998), and these
processes make them excellent candidates for the phytoremediation of
Se-contaminated sites (Terry and Zayed, 1994, 1998; Zayed and Terry,
1994; Banuelos et al., 1995). Phytoextraction is an important
remediation process by which plants remove trace elements (such as Se)
from contaminated environments and absorb them into their tissue
(Kumar et al., 1995). The plant tissue can be harvested, thereby
removing the Se from the site. Phytovolatilization is also an important
remediation process because it prevents Se from entering the food
chain. Atmospheric studies on the fate of dimethylselenide show that
the volatile Se is deposited away from the contaminated site in soils
that might be deficient in Se (Atkinson et al.,
1990).
A laboratory screening study conducted with 20 crop species identified
Indian mustard (Brassica juncea L.) as one of the best plant
species for Se phytoremediation because it grows rapidly, produces a
large biomass, and efficiently volatilizes and accumulates Se (Terry et
al., 1992; Terry and Zayed, 1998). Compared with the other species
tested, Indian mustard was the most efficient in extracting inorganic
Se from hydroponic solution. Some of the extracted Se was
bioconcentrated in plant tissues and some was volatilized. These
laboratory data are consistent with results from field trials that
showed that Indian mustard efficiently removed Se from
selenate-contaminated soil (Banuelos and Meek, 1990; Banuelos et al.,
1993), with up to 50% of the soil Se removed from a depth of
0 to 75 cm during a 3-year period (Banuelos et al., 1995).
Se-volatilization rates from this plant species have not been measured
in the field.
In addition to Se volatilization by plants, the production of volatile
Se has been measured from algae, fungi, and bacteria, and from samples
collected from upland and wetland environments (this literature was
recently reviewed by Terry and Zayed [1998] and Frankenberger and
Karlson [1994]). Experiments with bactericide and fungicide
amendments to wetland sediment-enrichment cultures showed that bacteria
were more important than fungi in Se volatilization in wetlands
(Azaizeh et al., 1997). Enrichment cultures of wetland sediment
rhizosphere microbes volatilized Se at higher rates than bulk sediment
microbes. This is because there are greater numbers of microbes and
higher microbial activity in the rhizosphere than in bulk soils and
sediments (Anderson et al., 1993; Sorensen, 1997). Because
microbes in the rhizosphere interact with plants by affecting plant
growth, enhancing mineral and water uptake, producing antibiotics to
inhibit soil pathogens, and producing plant-growth regulators
(Kapulnik, 1996), it is possible that they influence Se assimilation by
plants and, therefore, affect Se phytoremediation.
Preliminary data have shown that bacteria may increase the ability of
plants to volatilize Se. The rate of Se volatilization from selenate
was higher from detopped broccoli plants (plants with shoots that had
been removed) than from intact plants (Zayed and Terry, 1994). When
bactericides were added to the nutrient solution of these detopped
plants, the rate of Se volatilization was inhibited, presumably because
the bacteria in the rhizosphere of the detopped plants were inhibited.
Cycloheximide, a fungicide, did not have any effect on the rate of Se
volatilization, suggesting that fungi were not involved in Se
assimilation and volatilization by these plants. Therefore, the
objective of this study, using Indian mustard as a model species, was
to determine the role of rhizosphere bacteria in Se accumulation and
volatilization by plants.
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MATERIALS AND METHODS |
Antibiotic Experiments
Indian mustard (Brassica juncea L.) seeds (accession
no. 173874) were obtained from the North Central Regional Plant
Introduction Station (Ames, IA). Seeds were germinated on moistened
filter paper and transferred after 2 d into 4-inch pots containing
coarse sand. The pots were maintained in a greenhouse with controlled temperature (25°C-30°C) and a short-day (9 h) photoperiod. The plants were watered twice a day, once with tap water and once with
half-strength Hoagland solution (Hoagland and Arnon, 1938). After 1 month the plants were carefully removed from the sand to avoid damage
to the roots, washed in deionized water, and placed in plastic
containers containing half-strength Hoagland solution. The hydroponic
solutions were aerated. After 1 week in hydroponic solution the plants
were transferred into solutions containing 0.1 mg
mL
1 ampicillin (to inhibit bacteria) and 20 µM Se supplied as sodium selenate. Ampicillin
inhibits both gram-positive and gram-negative bacteria; it was the
antibiotic of choice because it interferes with bacterial cell wall
synthesis and is therefore expected to have the least effect on plant
physiology. For use as controls, another set of plants was maintained
in Se-containing hydroponic solution without ampicillin.
After 1 week on Se, the hydroponic solution was replaced with fresh
half-strength Hoagland solution with or without ampicillin (depending
on the treatment) and 20 µM selenate. Se volatilization was measured for a 24-h period as described below. Microbial numbers were estimated in 50-mL samples of nutrient solution from
ampicillin-treated and untreated plants by acridine orange direct
counts (Hobbie et al., 1977). The plants were then washed thoroughly in
running water to remove any Se that may have adhered to the roots,
dried at 70°C for 3 d, and then weighed. Roots and shoots were
ground separately using a Wiley mill, and Se analysis in the tissues was carried out by acid digestion (Martin, 1975) followed by
vapor generation-atomic absorption spectroscopy (Logan et al., 1987). The detection limit of Se in this analytical method was 1 µg
L1. A wheat flour standard (Se at 1.1 mg
kg1) and a blank were used with all digestions.
Se dioxide reference solution (Fisher) was diluted in 6 M
HCl and used as a standard. All samples were diluted in 6 M
HCl to give absorbances in the linear portion of the standard curve.
Three replicate plants were used for each treatment in all experiments.
Statistical analyses were performed using the JMP IN statistical
package (SAS Institute, Cary, NC) using analysis of variance
procedures.
Se-Volatilization Measurements
Se volatilization was measured by placing the plants in Magenta
boxes (Sigma) containing half-strength Hoagland solution and 20 µM selenate. The Magenta boxes were placed in gas tight
acrylic volatilization chambers (3-L volume) through which a continuous air flow (1.5 L min1) was passed by applying
suction at the outlet and by bubbling incoming air into the hydroponic
solution. Volatile Se was quantitatively trapped in alkaline peroxide
liquid traps as described previously (Zayed and Terry, 1992). For the
axenic plant experiments, the chambers were disinfected with 20%
bleach, sterile hydroponic solutions were used, and sterile air
(0.22-µm-filter sterilized) and sterile tubes were used to aerate the
hydroponic solution. The Se-volatilization chambers were placed in a
plant growth chamber with a 24-h photoperiod and maintained at 25°C
with an irradiance of 400 µmol m2
s1 photosynthetic photon flux (mainly as
fluorescent light with some incandescent light). Aliquots of trap
solution were kept at 4°C until analysis. The trap-solution samples
were heated at 95°C to remove the peroxide. The selenate-Se was
reduced to selenite by adding an equal volume of concentrated HCl and
heating at 95°C for 30 min. The Se concentration was measured by
vapor-generation atomic absorption spectroscopy as described above.
Isolation of Bacteria
Indian mustard plants were grown in soil and then transferred to
hydroponic medium containing 20 µM selenate as described above. The plant shoots were detopped and individual roots were rinsed
in sterile water. Excess water was removed by blotting the roots on
sterile filter paper. The plant roots were then inserted (using sterile
forceps) into tubes containing a basal salts medium (de Souza and Yoch,
1995) of 0.3% agar; the medium was similar to that described earlier
except that it contained 20 µM selenate and 5 mM acetate instead of acrylate as the C source. Se was used in the medium to obtain Se-tolerant bacterial isolates. The tubes were
incubated in the dark at room temperature for 3 d, after which
bacterial colonies were visible in the medium next to the roots.
Phenotypically different bacterial colonies were picked out of the
medium using sterile inoculation needles and streaked out on plates
containing the same Se-containing basal salts medium solidified with
1.5% agar. These bacteria, which also grew on tryptic soy agar plates,
can be characterized morphologically and phylogenetically.
Axenic Plants and Bacterial Inoculations
Indian mustard seeds were surface-sterilized by treatment with
70% ethanol for 30 s, 20% hypochlorite for 30 min, and five washes with sterile water. During these treatments the seeds were kept
in tightly closed, sterile, plastic tubes and kept on a rocking platform to ensure equal access of the bleach and alcohol to all of the
seeds. After the seeds were blotted on filter paper in a laminar flow
hood, they were transferred into sterile double Magenta boxes
containing 200 mL of 0.22-µm-filter sterilized half-strength Hoagland
solution. The Magenta boxes contained sterile wire mesh grids that were
made to perfectly fit the sides of the box and just touch the surface
of the hydroponic solution. A small square was cut in one corner of the
grid and a Nalgene (Milwaukee, WI) tube was inserted through it
so that the nutrient solution could be aerated during the
Se-volatilization measurements. Fifty seeds were placed on each grid
and allowed to germinate. The seedlings were kept in a growth chamber
maintained at the same conditions as the Se-volatilization chambers
(see above) for 1 month.
For inoculation of axenic plants with bacteria to determine the optimum
plant-microbe combinations for Se accumulation and Se volatilization,
pure cultures of all bacterial strains used in this study were grown in
tryptic soy broth for 18 h on a shaker at 200 rpm. The only
exception was strain BJ2, which was grown for 36 h because of its
slow growth rate. The cultures were centrifuged at 8000g for
10 min. The pellet was resuspended in 50 mM
phosphate buffer, pH 7.0, recentrifuged, and finally resuspended in 5 mL of phosphate buffer. Equal numbers of bacteria (approximately 104 colony-forming units
mL1) were added to the rhizospheres of axenic
plants maintained in hydroponic solution containing 20 µM selenate. The number of viable bacterial
cells was measured by serial dilution in sterile 0.85% NaCl, followed
by viable counts on tryptic soy agar plates. After a 7-d treatment with
Se and bacteria, the plants were rinsed in sterile water and placed in
fresh, sterile hydroponic solution containing 20 µM selenate to minimize the contribution of
bacteria to volatile Se production. After 7 d the bacteria were
assumed to colonize the root, and therefore, no further addition of
bacteria was made when the hydroponic solution was replaced. Procedures described above for the antibiotic experiments were used to measure tissue Se concentration and the rate of Se volatilization from axenic
plants and from axenic plants to which bacteria had been added.
Controls for the Se-volatilization experiment from axenic plants
inoculated with strain BJ2 or BJ15 were set up as follows. Strains BJ2
and BJ15 were grown in tryptic soy broth and washed as described above.
Different volumes (0.1-1 mL) of the washed cell suspensions were added
to 200 mL of half-strength hydroponic solution with 20 µM
selenate to generate samples with different bacterial cell numbers, and
Se-volatilization rates were measured for a 24-h period using the same
incubation conditions as for plants. Se-volatilization rates were
linearly dependent on the bacterial cell numbers (data not shown).
After measuring the rate of Se volatilization from plants that had been
inoculated with strain BJ2 or BJ15, bacterial cell numbers were
estimated in the nutrient solution, and the rate of Se volatilization
for that number of bacteria was estimated from the curve of
Se-volatilization rate versus bacterial cell numbers. The amount of Se
volatilized during a 24-h period by strains BJ2 and BJ15 alone was
subtracted from that volatilized by the plant-bacterial combination
during the same period.
Root Exudate Experiments
The hydroponic solution that was used to culture the plants was
considered to contain diluted root exudate. The root exudate was
filtered through a 0.22-µm membrane to remove any bacteria present in
the solution. The amino acid content of bacteria-free root exudate was
determined by phenol:HCl hydrolysis followed by ion-exchange
chromatography with a sodium citrate buffer on an amino acid analyzer
(model 6300, Beckman) at the University of California, Davis, Protein
Structure Laboratory. The protein content of root exudate was estimated
using the Bradford procedure (Bio-Rad) and BSA as a standard.
To determine if bacteria cause the production of a heat-labile
bioactive compound in root exudate, and to determine if this compound
was involved in enhancing Se accumulation in plants, 75 mL of
filter-sterilized root exudate from axenic and inoculated plants was
boiled for 5 min or left untreated, and then added to new batches of
axenic plants growing in sterile Magenta boxes containing 75 mL of
sterile, half-strength Hoagland solution. Selenate was added at 20 µM, and the Se content of tissues was measured after 1 week, as described above.
Effect of Bacteria and Se on Plant Growth
Indian mustard seeds were surface-sterilized as described above.
Sterilized seeds were blotted dry on sterile filter paper in a laminar
flow hood. The seeds were then allowed to soak for 20 min in a sterile
0.5% methylcellulose solution that did or did not contain a pure
culture of bacterial cells at a density of 108
colony-forming units mL1. The seeds were
removed from the methylcellulose solution containing bacteria and dried
on a sterile paper towel placed in a laminar flow hood. Fifty seeds
were transferred aseptically into sterile Magenta boxes containing 50 mL of autoclaved half-strength Murashige and Skoog medium
(without Suc) that had been amended with 0, 20, 50, 100, or 250 µM Se as sodium selenate. The selenate was filter sterilized through a 0.22-µm filter and added to the medium before it
solidified. The Magenta boxes were incubated in a growth chamber maintained at 25°C, 40% humidity, and constant light. After 1 week
plants were removed from the agar medium and the length of the longest
root and the fresh weights of the seedlings were measured. Some
seedlings were processed for microscopy as described below. The
remaining seedlings were carefully washed to remove any agar, digested,
and analyzed for their Se content, as described above.
To determine the effect of bacteria on root morphology, both axenic and
bacteria-inoculated seedlings were fixed in 0.05% Tween 20 for 10 min,
washed with water, stained with a 105 dilution
of acridine orange for 10 min, and washed for 10 min in water. The
acridine orange-stained samples were optically sectioned using the 10×
objective (0.45 numerical aperture) of a confocal laser scanning
microscope (Sarastro 1000, Molecular Dynamics, Sunnyvale, CA) with an
Ar ion laser (excitation, 488 nm; emission, 525 nm). Image files
were acquired and three-dimensional projections were rendered on an SGI
Indy R 4400 using ImageSpace 3.2 (Molecular Dynamics). The
resulting images were saved as TIFF files for use in Adobe Photoshop
for Macintosh (Apple). The same samples were viewed using a
fluorescence microscope (Zeiss Axiophot) with a HBO 100-W mercury light
source and a 5× objective. Samples were excited using a 450- to 490-nm
excitation filter and viewed with a 520-nm emission filter. Images were
captured using a color video camera (Optronics 450, Optronics
International, Chelmsford, MA) and a Scion C67 frame-grabber board on a
Macintosh computer using Scion Image (color NIH Image v. 1.62). All
images were saved as TIFF files for Adobe Photoshop for Macintosh.
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RESULTS |
Use of Antibiotic Experiments to Determine the Role of Bacteria in
Se Accumulation and Volatilization
Our results suggest that bacteria in the rhizosphere facilitate Se
accumulation and volatilization by plants. The rate of Se
volatilization from ampicillin-supplied plants (with inhibited rhizosphere bacteria) was significantly lower than that of plants with
their naturally occurring rhizosphere bacterial populations (P < 0.05; Fig. 1A). Furthermore, the Se
concentrations in roots and shoots of ampicillin-treated plants were
lower than those in untreated plants (P < 0.05; Fig. 1B).
Ampicillin added to the nutrient solution of plants inhibited Se
volatilization by about 35% and tissue Se accumulation by about 70%.
It is evident that ampicillin inhibited the growth of bacteria because
acridine-orange direct counts of total microbes in the nutrient
solution of ampicillin-treated plants were 4-fold lower than microbe
counts of plants without ampicillin (data not shown).
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| Figure 1.
Role of bacteria in Se volatilization (A) and
accumulation (B) from plants supplied with 20 µM
selenate. Ampicillin at 100 mg L1 was used as an
antibiotic to inhibit rhizosphere bacteria. The mean and SD
values of three replicates are shown. Differences in Se accumulation
and volatilization between ampicillin-treated and untreated plants were
significant (P < 0.05). DW, Dry weight.
|
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Use of Axenic Plant Experiments to Determine the Role of Bacteria
in Se Accumulation and Volatilization
Several strains of rhizosphere bacteria were isolated from Indian
mustard plants grown in soil to determine the effect of pure cultures
on Se accumulation into plant tissues. When individual strains of
bacteria isolated from Indian mustard (Fig.
2, the BJ strains) were added to the
rhizosphere of axenic Indian mustard plants supplied with selenate,
most of the bacterial isolates significantly enhanced the ability of
axenic plants to accumulate Se in their roots and shoots (P < 0.05). One of the rhizosphere bacterial strains (BA5), which was
isolated from salt-marsh bulrush (Scirpus robustus) roots,
also enhanced Se accumulation in Indian mustard. Therefore, the
rhizosphere bacteria are not plant specific in enhancing tissue Se
accumulation. Different bacterial strains had different abilities to
enhance Se accumulation in plants. For example, strains BJ1 and BJ2
were much better than strain BM3 at enhancing Se uptake into axenic
Indian mustard.
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| Figure 2.
Inoculation of bacterial isolates into the
rhizosphere of axenic plants leads to increased Se accumulation in
shoots (A) and roots (B) of plants supplied with 20 µM
selenate. The mean and SD values of three replicates are
shown. All bacterial strains were isolated from the rhizosphere of
Indian mustard plants except for strains BM3 and BA5, which were
isolated from the rhizosphere of salt-marsh bulrush plants. The
differences in tissue Se accumulation for axenic plants compared with
plants inoculated with bacteria were significant in most cases (P < 0.05). The following bacteria-treated plants showed nonsignificant
differences from axenic tissue Se accumulation: shoot Se accumulation
for strains BJ13, BJ6, BJ11, and BJ15, and both root and shoot Se
accumulation for strain BM3. DW, Dry weight.
|
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In addition to facilitating Se accumulation into plant tissues,
rhizosphere bacteria also enhanced Se volatilization by plants (Fig.
3). Bacterial strains BJ2 and BJ15 were
identified as good candidates for enhancing Se accumulation into plants
(Fig. 2). When these bacterial strains were inoculated into the
rhizosphere of axenic Indian mustard plants, the rate of Se
volatilization from selenate was 4-fold higher than that of axenic
control plants (P < 0.05; Fig. 3A). Furthermore, similar to the
data presented in Figure 2, plants inoculated with strains BJ2 and BJ15
accumulated more Se in their tissues (Fig. 3C); compared with axenic
controls, both bacterial strains increased the Se concentrations in
shoots and roots 1.4- and 5-fold, respectively (P < 0.05). The
protein content of bacteria-free root exudate was also significantly
increased in plants with rhizosphere bacteria compared with axenic
plants (P < 0.05; data shown in the legend to Fig. 3B).
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| Figure 3.
Inoculation of bacterial strains BJ2 and BJ15 into
the rhizosphere of axenic plants leads to increased Se volatilization
(A). Inoculation of these bacteria into the rhizosphere of axenic
plants also increases the protein content of the root exudate (B) and
Se accumulation in plant tissues (C). Bacterial strains BJ2 and BJ15
were identified as superior strains for plant tissue Se accumulation
(Fig. 2). The mean and SD values of three replicates are
shown. Differences in Se accumulation and volatilization between
bacteria-treated and untreated plants were significant (P < 0.05). The amount of Se volatilized during a 24-h period by strains BJ2
and BJ15 alone (0.08 and 0.37 µg d1) was subtracted
from the amount volatilized by the plant-bacteria combination during
the same period. DW, Dry weight.
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Effect of Bacteria and Se on Plant Growth
Initially, bacteria were thought to enhance Se uptake into Indian
mustard by stimulating root-hair production. Plants that germinated in
media containing 20 µM selenate from seeds coated with
bacteria had more and longer root hairs than plants that germinated
from axenic seeds (Fig. 4). Compared with
axenic plants, root-hair production in bacteria-supplied plants began
closer to the root apical meristem. Scanning electron micrographs
showed that bacteria colonized all areas of the roots of seedlings that germinated from bacteria-coated seeds; no bacteria were detectable on
axenic seedlings (data not shown). As the Se concentration in the
medium increased from 0 to 20 µM, the length of the root hairs of bacteria-supplied plants appeared to be unaffected by Se,
whereas that of axenic plants decreased. At Se concentrations of 50 µM or higher, plants with bacteria still had increased
root surface area compared with axenic controls, although the roots and
root hairs were much shorter (data not shown).
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| Figure 4.
Axenic plants infected with bacterial strain BJ2
(A and C) had increased root-hair production compared with axenic
plants (B and D) supplied with 0 and 20 µM selenate in
agar medium. Seedlings were germinated from surface-sterilized seeds
coated with or without bacteria in a methylcellulose paste, stained
with acridine orange, and observed at 5× magnification. Similar
results were observed when strain BJ15 was used and when the root tips
were observed at 10× magnification by confocal microscopy. Bars = 100 µm.
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As the Se concentration in the medium increased, there was a decrease
in the root length of seedlings with or without bacterial strains BJ2
or BJ15 in their rhizosphere. Seedlings that germinated from seeds
coated with bacterial strain BJ2 or BJ15 had significantly shorter
roots than axenic seedlings at most external Se concentrations (P < 0.05; Fig. 5A); at 250 µM there was no significant difference in root length
between seedlings with and without bacteria. The fresh weights of
seedlings with and without bacteria were similar and decreased with
increasing Se concentrations in the growth medium (Fig. 5B). At a Se
concentration of 250 µM, the seedlings that germinated
from bacteria-coated seeds had significantly higher fresh weights than
axenic seedlings (P < 0.05). The seedlings with bacteria in their
rhizosphere had significantly higher tissue Se concentrations (1.3- to
2-fold) compared with axenic plants at all external Se concentrations
tested (P < 0.05; Fig. 5C).
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| Figure 5.
Effect of bacteria and different Se concentrations
on growth and Se accumulation by axenic plants grown in agar. A, The
length of the longest root; B, fresh weight of individual seedlings;
and C, Se concentration in seedling tissue (root plus shoot). All
differences between bacteria-treated and untreated axenic plants were
statistically significant (P < 0.05) except for root lengths at
250 µM Se and fresh weights at 0, 20, and 100 µM Se. DW, Dry weight.
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Experiments with Root Exudate
Compared with axenic plants, plants with bacteria in their
rhizosphere had increased levels of amino acids in their growth medium,
which was considered as diluted root exudate and had been filter
sterilized to remove the bacteria (Fig.
6). The concentration of all of the amino
acids measured was at least 2-fold higher in the root exudate of plants
with bacteria compared with that of axenic plants, and Ser levels were
even enhanced 9-fold. The total protein content of the
filter-sterilized root exudate from plants with bacteria was 2.8-fold
higher than that of plants without bacteria (Fig. 6, inset).
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| Figure 6.
The amino acid and protein contents of 0.22-µm
filtered root exudate from plants inoculated with strain BJ2 were
higher than those from axenic plants; all were supplied with 20 µM selenate. The amino acid concentration in root exudate
from bacteria-supplied plants (0.5-23.6 nmol mL1) was
divided by that of axenic plants (0.2-9.2 nmol mL1). The
protein content shown in the inset was calculated from the amino acid
content.
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To determine if a bioactive compound in the root exudate of the
plant-bacterial combination facilitated Se accumulation into plant
tissues, bacteria-free root exudate from a plant-microbe combination
was either left untreated or boiled, and then added to a fresh batch of
axenic plants. This bacteria-free root exudate from a plant-microbe
combination enhanced Se accumulation in axenic plant roots 5.5-fold
(P < 0.05; Fig. 7). Boiled root
exudate had no such effect. Se accumulation in shoots was 2-fold higher
in plants that received root exudate from a plant-bacteria combination compared with the axenic controls (data not shown); however, this difference was not statistically significant. The negative control for
this experiment was untreated or boiled root exudate from axenic plants
added to a fresh batch of axenic plants. The negative controls did not
have higher tissue Se concentrations than axenic plants (compare Fig.
7, bars on left, and Fig. 2, first bar). When root exudate was
concentrated 1000-fold by freeze drying, resuspended in buffer,
dialyzed, and analyzed by native and SDS-PAGE, no proteins in the
exudate were visible on the gel.
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| Figure 7.
The bacteria-plant combination produced a
heat-labile compound in diluted root exudate that, when supplied to
axenic plants, enhanced the Se concentration in roots 5-fold. The mean
and SD values of three replicates are shown. Differences in
root Se concentrations of axenic plants treated with growth medium
(diluted root exudate) supplied with bacteria and axenic plants treated
with root exudate or boiled exudate were significant (P < 0.05).
The root exudate from axenic plants or plants grown with strain BJ2 was
filtered through a 0.22-µm filter to remove bacteria, and then added
to a fresh batch of axenic plants with 20 µM selenate.
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DISCUSSION |
This study showed that bacteria in the rhizosphere are necessary
to achieve optimum rates of Se accumulation and volatilization by
plants. The rate of Se accumulation and volatilization from plants is
also dependent on the chemical form of Se, competition due to sulfate,
concentration of Se, the plant species, the age of the plant, and other
environmental factors such as pH and temperature (Terry et al., 1992;
Zayed and Terry, 1992; Terry and Zayed, 1994; de Souza et al., 1998;
Pilon-Smits et al., 1998). Recent kinetic studies have shown that
selenate uptake and maintenance in the root may be rate limiting for Se
assimilation and volatilization by Indian mustard (de Souza et al.,
1998). The present study showed that rhizosphere bacteria can overcome
part of the rate limitation, i.e. Se uptake, and thereby increase Se
accumulation in tissues and Se volatilization. The reduction of
selenate inside the plant was also shown to be rate limiting for Se
volatilization (de Souza et al., 1998). Bacteria in the rhizosphere
should have little effect on Se reduction within plant tissue, and
should not, therefore, have as great an effect on Se volatilization as
on Se uptake. This contention is supported by the results from the
antibiotic experiment (Fig. 1), which showed that bacteria played a
larger role in plant Se accumulation (70%) than in volatilization
(35%). This disproportional increase in tissue Se accumulation
compared with Se volatilization may also be attributable to the rapid
translocation of selenate into shoots (de Souza et al., 1998; Zayed et
al., 1998) away from the root, which is the site of volatilization (Zayed and Terry, 1994).
The bacterial strains used in this study were isolated from roots and
selected from the general rhizosphere population based on their ability
to tolerate 20 µM Se; therefore, the isolates obtained
were very likely to be involved in Se transformations in the
rhizosphere. Indeed, the ability to increase plant tissue Se
accumulation and volatilization was a characteristic shared by most of
these Se-tolerant bacterial strains (Fig. 2); strain BM3 was an
exception. The Se-tolerant bacterial strains were not plant specific;
for example, strain BA5, which was isolated from the rhizosphere of
bulrush, also enhanced Se accumulation in Indian mustard. Similar
results were found with axenic broccoli (Brassica oleracea
botrytis L.) plants, in which rhizosphere bacteria isolated from
Indian mustard or salt-marsh bulrush also enhanced Se uptake. This
information is beneficial because it shows that a bacterium from a
certain plant species may be used in conjunction with other plant
species to improve their phytoremediation potential. Many of the
bacteria tested in this study can also accumulate, precipitate, and
volatilize Se on their own (M. de Souza, D. Chu, M. Zhao, and N. Terry,
unpublished). The contribution of bacteria to Se volatilization (when
nonaxenic plants were used) was minimized by rinsing the plants in
sterile water to remove loosely bound bacteria from the roots before
the Se-volatilization measurements, and by using controls to estimate
the rate of Se volatilization by bacteria alone.
Bacteria in the rhizosphere interact in numerous ways with plants to
improve their growth (Kapulnik, 1996). Aside from the well-characterized nitrogen-fixing legume symbioses and plant-pathogen host interactions, rhizosphere bacteria can stimulate plant growth by
producing phytohormones (Fallik et al., 1994), enhancing mineral and
water uptake (Lin et al., 1983), producing antibiotics to inhibit
pathogens (Lesinger and Margraff, 1979), and altering root morphology
(Lin et al., 1983; Kapulnik, 1996). The present study showed that
bacteria appear to increase the plant's potential for Se
phytoremediation because they facilitate Se accumulation and
volatilization.
In the presence of high Se concentrations, rhizosphere bacteria caused
plants to increase their root-hair production (Fig. 4). When
rhizosphere bacteria were inoculated onto surface-sterilized Indian
mustard seeds and the seeds were germinated in tissue-culture medium,
the bacteria colonized the roots and enhanced their surface area.
Initially, it was assumed that the increased root surface area of
plants with bacteria caused their increased tissue Se uptake and
accumulation. However, when other chemical forms of Se, selenite and
SeMet, were supplied to plants, the tissue Se concentrations were not
enhanced in plants inoculated with bacteria compared with axenic plants
(data not shown). Therefore, it is unlikely that the stimulation of
root-hair production by bacteria is responsible for enhancing Se
accumulation from selenate.
A heat-labile compound was shown to enhance Se accumulation in axenic
plants. This compound has not yet been identified, and it is not clear
whether it is produced by the plant or by the rhizosphere bacteria.
Because the protein content of root exudate from plants with bacteria
in their rhizosphere was higher than that of axenic plants (Figs. 3 and
6), and because these plants also had higher rates of Se accumulation
and volatilization (Fig. 3), the heat-labile compound could be
proteinaceous in nature and could also enhance Se volatilization. The
root exudate of axenic plants inoculated with bacteria had a higher
amino acid content than that of axenic plants; in particular, Ser
levels were greatly enhanced. It is possible that adding Ser or its
derivatives to axenic plants will enhance Se volatilization because
o-acetylserine is an intermediate in the proposed
Se-volatilization pathway (Terry and Zayed, 1994), where it is involved
in the incorporation of inorganic Se into selenocysteine
The heat-labile compound produced by the plant-bacteria interaction may
be involved in stimulating the selenate transporter. Alternatively, the
heat-labile compound produced by the plant-bacteria interaction may be
a reductant or enzyme produced by the bacteria, which is capable of
converting selenate to organic Se compounds such as SeMet. Earlier
studies with Indian mustard and broccoli plants have shown that plants
take up SeMet at faster rates than selenate, and that the root-to-shoot
ratio for tissue Se concentration is higher when SeMet is supplied,
compared with selenate (Zayed et al., 1998). Figure 3 provides some
evidence to support the contention that some of the bacterial strains
used in this study may enhance selenate uptake by converting selenate
to organic Se in the rhizosphere. Compared with axenic plants, plants
supplied with bacterial strains BJ2 and BJ15 had 4- to 6-fold higher
root Se concentrations (consistent with faster rates of uptake into roots), and 3- to 4-fold higher root-to-shoot ratios for tissue Se
concentration, consistent with lower rates of translocation from root
to shoot, a characteristic shown by SeMet-amended plants (Zayed et al.,
1998). It is unlikely that bacterial reduction of selenate to selenite
(Macy et al., 1993; Oremland et al., 1994; Losi and Frankenberger,
1997) contributes greatly to the enhanced Se uptake in
bacteria-supplied plants treated with selenate, compared with axenic
plants. This is because selenite is taken up at slower rates than
selenate, which, in turn, is taken up at a slower rate than SeMet (de
Souza et al., 1998; Zayed et al., 1998).
The fact that bacteria only enhance Se accumulation in plant tissues
when supplied with selenate (but not selenite or SeMet) is interesting
because selenate is the major form of Se in soils in the San Joaquin
Valley and in agricultural drainage water resulting from irrigation
(McNeal and Balisteri, 1989). Furthermore, this result suggests that
different uptake mechanisms exist for the different chemical forms of
Se. Selenate, a chemical analog of sulfate, is thought to be taken up
into plants by active uptake via the sulfate transporter protein
located in the root plasma membrane (Leggett and Epstein, 1956),
selenite is thought to be taken up passively (Arvy, 1993), whereas
SeMet is thought to be taken up actively in a manner similar to that of
S-containing amino acids (Sandholm et al., 1973; Abrams et al., 1990).
The results from the present study suggest that bacteria in the
rhizosphere may stimulate only the sulfate transporter protein and not
the SeMet transporter protein. This would explain why bacteria
stimulate only selenate accumulation and not selenite or SeMet
accumulation in plants (data not shown).
Bacteria that were identified in the laboratory as being superior for
enhancing Se phytoextraction and phytovolatilization (Figs. 2 and 3)
could be tested under field conditions. For example, germinating
bacteria-coated seeds in Se-contaminated soil could make the
phytoremediation of selenate-contaminated sites more efficient if the
selected bacteria can compete favorably with the resident populations.
In addition to being an excellent species for Se phytoremediation
(Banuelos and Meek, 1990; Banuelos et al., 1995; Zayed and Terry,
1998), Indian mustard is an excellent candidate for the
phytoremediation of metals from contaminated soil and water through
processes such as phytoextraction and rhizofiltration (Dushkenov et
al., 1995; Kumar et al., 1995). Also, Indian mustard used in
conjunction with rhizosphere bacteria that are superior at enhancing
plant Se accumulation and volatilization may be used to remove Se from
contaminated soils and agricultural drainage water in the San Joaquin
Valley and other places where Se contamination is a problem.
| |
FOOTNOTES |
1
This work was supported by the Electric Power
Research Institute (grant nos. W08021-30 and W04163 to N.T.).
*
Corresponding author; e-mail nterry{at}nature.berkeley.edu; fax
1-510-642-3510.
Received June 29, 1998;
accepted October 14, 1998.
| |
ABBREVIATIONS |
Abbreviation:
SeMet, selenomethionine.
| |
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Top
Abstract
Introduction
