RESEARCH FRONT
Rapid Communication
CSIRO PUBLISHING
www.publish.csiro.au/journals/env
G. Evans et al., Environ. Chem. 2005, 2, 167–170. doi:10.1071/EN05046
Unexpected Beneficial Effects of Arsenic on Corn Roots
Grown in Culture
Grant Evans,A Julyette Evans,B Andrea Redman,C Nancy Johnson,C,D
and Richard D. Foust, Jr.A,C,D,E
A
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011, USA.
for Sustainable Environments, Northern Arizona University, Flagstaff, AZ 86011, USA.
C Center for Environmental Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA.
D Merriam–Powell Center for Environmental Research, Northern Arizona University, Flagstaff, AZ 86011, USA.
E Corresponding author. Email: Richard.Foust@nau.edu
B Center
Environmental Context. Phytoremediation, the process of using plants to remove metals from contaminated soils, shows promise as a low-technology method for economically removing arsenic, and other toxic
metals, from soil. Arsenic transport studies in vascular plants have examined how arsenic is taken up, chemically modified, and transported from roots to other parts of the plant. No studies, to our knowledge, have
examined the effect of low-level doses of arsenic on the roots themselves. This paper shows, for the first
time, that arsenic at low levels may beneficially affect root development.
Abstract. Corn (Zea mays) roots were grown in culture on modified Strullu–Roman medium in two separate
experiments. Roots were exposed to one of four treatments combining arsenic (100 µg L−1 or 0.0 µg L−1 ) and
phosphorous (4.8 mg L−1 or 0.0 mg L−1 ). The cultures were allowed to grow for 18 days or 21 days before they
were used for quantitative measurement of root mass, root length, number of branches, and branch length. Results
indicate roots grown in medium lacking phosphate but containing arsenic were longer and had greater mass than
roots grown in medium with only phosphate. The data presented here suggest that arsenic at low levels might be
beneficial for root development.
Keywords.
arsenic — biological monitoring (plants) — contaminant uptake — phytoremediation
Manuscript received: 12 June 2005.
Final version: 15 August 2005.
Arsenic is a naturally occurring element that is prevalent in
crustal soils at low abundance, and often associated with iron
mineral complexes, sulfates and ores like copper, lead, and
gold.[1] Arsenic can exist in four oxidation states (−3, 0, +3,
+5), but in the environment arsenic is most often found in
either as arsenite (AsIII ; H3AsO03 and H2AsO−
3 ) or arsenate
2− [2]
(AsV ; H2AsO−
and
HAsO
).
The
2001
report
of arsenic
4
4
hyperaccumulation by the Chinese Brake Fern (Pteris vittata L.)[3] resulted in a race to understand arsenic uptake by
P. vittata and to identify other plants that may have similar
properties.[4–6] Little data exists, however, of the low-level
effects of arsenic on root development.
Arsenate has been shown to act as a phosphate
analogue,[7,8] where an organism will assimilate arsenate as
it would phosphate. Arsenic is prevalent in many surface
and sub-surface waters, causing poisoning and health problems when arsenic-contaminated water is used for irrigating
crops. Two recent studies document the movement of arsenic
from soil and groundwater into the food chain. Islam et al.[9]
recently reported arsenic levels as high as 2.05 mg kg−1
© CSIRO 2005
167
for rice grains collected from Bangladesh, where the crops
were grown with arsenic-contaminated irrigation water. In a
greenhouse study Abedin et al.[10] demonstrated that arsenic
concentrations increased in the roots, straw, husk, and grain
of rice grown in water containing arsenic in concentrations
of 0 to 8 mg L−1 . Other studies have investigated heavy metal
uptake by vegetables grown on highly contaminated soils.[11]
A quantitative study reporting the effects of low-level arsenic
concentrations on root tissue in pure culture has not been
reported.
Corn (Zea mays) seeds (Johnny Seed, F1 Yellow Select)
were surface-sterilized using a series of washe and rinse steps.
First, the seeds were soaked for 2 min in a 10% solution of
household bleach (3% w/v sodium hypochlorite) then rinsed
five times in distilled, deionized water. Second, the seeds were
soaked with 95% ethanol for one minute before five more
rinses with sterile, distilled, deionized water.
Following the final rinse, seeds were transferred to
sterile 150 mm plastic disposable Petri plates. These Petri
plates had been previously prepared by placing four sheets
1448-2517/05/030167
RESEARCH FRONT
G. Evans et al.
Table 1. Reagent concentration in medium
Reagent
MgSO4 ·7 H2 O
KNO3
KH2 PO4
Ca(NO3 )2 ·4 H2 O
Na2 Fe EDTA
KCL
MnCl2 ·4 H2 O
ZnSO4 ·7 H2 O
H3 BO3
CuSO4 ·5 H2 O
(NH4 )6 Mo7 O24 ·4 H2 O
Thiamine
Nicotinic acid
Pyridoxine
Calcium panthotenate
Biotin
Cyanocobalamin
Sucrose
Phytogel
Arsenic
Streptomycin
Stock concentration
[g L−1 ]
10
100
10
100
10
100
10
1
10
1
0.1
1
1
1
1
0.1
1
N/A
N/A
0.01
1
Target concentration
[mg L−1 ]
Quantity added
[mL]
74
76
4.1
359
8
65
2.45
0.29
1.86
0.24
0.035
1
1
0.9
0.9
0.009
0.4
10000
8000
0.1
100
of sterile number 40 filter paper dampened with 3 to
4 mL of sterile, distilled, deionized water, and 500 µL
of a sterile streptomycin (Sigma, 755 units mg−1 ) solution
(500 mg L−1 ). Seeds were placed in a single uniform layer
onto the moist filter paper. The Petri dishes were covered and
sealed in one-litre plastic zip-top storage bags.The seeds were
allowed to germinate in the dark for five days in a laminar
flow hood at 29.5◦ C.
Strullu–Roman medium was chosen for these experiments
because it is frequently used in root culture experiments
involving mycorrhizal fungi. Medium[12] was prepared by
adding aliquots of prepared stock reagent solutions to a
500 mL volumetric flask previously filled with 200 mL distilled, deionized water. The volumetric flask was heated and
stirred on a hot plate to ∼60◦ C during medium preparation.
Reagents were added in volumes to reach a target concentration (Table 1). Unless noted, reagents were obtained from
J. T. Baker Co. All reagents were prepared individually, except
for the solutions of thiamine (Fisher Scientific), nicotinic acid
(MP Biomedicals), pyridoxine (Acros Organics), and calcium
panthotenate (Acros Organics). Thiamine and nicotinic acid
were paired as were pyridoxine and calcium panthotenate.
The arsenic stock solution used (10 mg L−1 ) was prepared
by senal dilution of an ICP As standard (SPEX Standard,
1000 mg L−1 ).
Four medium compositions were created varying the relationship between arsenic and phosphorous in a full factorial
design (Table 2). In medium mixes where arsenic or phosphate were omitted those compounds were not added to the
medium in any amount. The flasks were lightly covered with
aluminum foil and autoclaved (liquid cycle at 121◦ C for
20 min) and allowed to cool in a laminar flow hood. Petri
dishes (60 mm) were filled with cooled medium to a capacity
of ∼2/3 by volume. The plates were covered and allowed to
set overnight, and then refrigerated at 38◦ C until needed.
3.695
0.380
0.205
1.795
0.400
0.325
0.123
0.145
0.093
0.120
0.175
0.500
0.500
0.450
0.450
0.045
0.200
5g
4g
2.5
50
Table 2. Arsenic and phosphate concentrations in the
medium used for this work
Treatment
As [µg L−1 ]
KH2 PO4 [mg L−1 ]
Medium 1
Medium 2
Medium 3
Medium 4
100
100
0
0
0
4.1
4.1
0
Germinated seeds were selected at random in groups of
four and transferred to a bleach-sterilized glass block. The
root sections were excised from the germ to a length between
15 and 18 mm. The cut sections were transferred singly to
previously prepared 60 mm Petri plates filled with medium.
Only one root section was allocated per plate. This procedure
was repeated until four experimental groups of four replicates were prepared. Each experimental group was sealed in
a litre-sized zip-top plastic bag for storage. Cultures were
maintained at 40◦ C in a laminar flow hood in sealed bags.
Storage bags were not opened and the cultures not disturbed
for 18 days.
Roots were removed from the culture medium and placed
into a 150 mm Petri dish that had been filled with a thin
(<1 mm) film of distilled, deionized water. The roots were
manually untangled with forceps and measured for length by
holding the uncoiled root against a calibrated stainless steel
ruler. The root was measured to the nearest millimetre. The
number of side branches were counted and the side branches
were measured to the nearest millimetre. Average side branch
length was determined by summing all side branch lengths
and dividing by total number of branches. Wet mass was
obtained by weighing (Ohaus AR2140d, ±0.001 g). Once all
the roots had been quantified, the four experimental groups
were pooled and air-dried to a constant mass. Individual root
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Beneficial Effects of Arsenic on Corn Roots
dry mass was calculated using the ratio of group dry mass to
group wet mass and multiplying the resulting factor by the
individual root wet mass.
Two solutions of dilute nitric acid (Fisher Scientific, Trace
Metal Grade) were prepared. A wash solution of 1.8% HNO3
was prepared by diluting 18 mL concentrated HNO3 in 1 L
distilled, deionized water. An ICP-MS reagent blank of 2.7%
HNO3 was prepared by diluting 27 mL HNO3 in 1 L distilled,
deionized water. A 100 µg L−1 solution of arsenic (SPEX
ICP-MS Standard, 1000 mg mL−1 ) was prepared using 2.7%
HNO3 as the diluent.
The arsenic solution used in these experiments was tested
for trace metal contamination, a possible cause of the
observed differences in root growth, by scanning the mass
spectrum for the presence of unintended metals between 20
and 150 amu. This was done using a VG Elemental AXIOM
multi-collector equipped inductively coupled plasma-mass
spectrometer (MC-ICP-MS). The instrument resolution was
manually set to 1000:1. The mass spectrum was recorded
for two solutions: (a) a 1.8% HNO3 solution containing
100 µg L−1 arsenic, and (b) a 1.8% HNO3 solution with no
arsenic. The mass spectrum for the blank solution was subtracted from the mass spectrum of the 100 µg L−1 arsenic
solution to identify metals present in the arsenic solution that
were not present in the blank. This experiment would have
identified any metals of masses 20 to 150, present at a concentration of 1 µg L−1 or greater, in the arsenic solution. No
trace impurities were found.
Root length, dry mass, degree of branching, and average
branch length were significantly affected by the addition of
arsenic to the medium (Table 3). ANOVA analysis of the
data from both studies, performed using JMP ver. 4.0.4,[13]
shows that adding 100 µg L−1 arsenic resulted in increased
root length and increased root mass. Both observations are
significant at α = 0.05 (Table 4). Although there were visible
changes to the root growth for the phosphate treatments, the
effects were much smaller than for arsenic and not statistically
significant.
Corn roots showed marked changes between the four treatments in this study (see Fig. 1). A clear pattern emerges;
both treatments where arsenic was added showed a marked
increase in root length, number of branches, and dry mass. All
response variables are highest in the treatments where arsenic
is added without phosphate and lowest when phosphorous but
not arsenic was added to medium.
Visually, the differences between the groups were telling.
Roots grown in medium with arsenic but without phosphate
were very different from roots grown in medium with phosphate alone. With no differences in medium synthesis or
processing, the only factor that could contribute to such a
difference is the arsenic concentration of the medium. The
pH was found to be constant among the medium groups and
was stable at 5.0.
The results of this study are contrary to what was expected.
Phosphate is an essential plant nutrient, but this study clearly
shows roots grown on medium containing a low concentration of arsenic surpass the growth of roots on medium with
only phosphate. Roots grown on medium lacking both arsenic
Table 3. Average of four samples from two root culture
experiments
Treatment
As+ P+
As+ P−
As− P+
As− P−
Study 1, 18 day exposure
Av. root length [mm]
Av. dry mass [mg]
No. of side branches
Av. branch length [mm]
115.5
3.5
21
32.3
129.2
3.7
37.5
41
25.0
1.1
0
0
47.2
2.3
18.25
8.8
Study 2, 21 day exposure
Av. root length [mm]
Av. dry mass [mg]
No. of side branches
Av. branch length [mm]
149.5
1.9
9.75
39.2
200.0
1.7
13.75
27.1
22.8
0.7
0
0
49.8
1.6
6.25
20.8
Table 4. ANOVA results for root cultures grown on medium with
different arsenic and phosphate treatments
Results that are significant at α = 0.05 are noted in bold
Effect
As
P
As×P
Average root length
(composite of both studies)
Average dry mass
(composite of both studies)
F = 48.24
P < 0.0001
F = 3.4847
P = 0.0724
F = 0.1428
P = 0.7084
F = 12.388
P = 0.0015
F = 2.223
P = 0.1466
F = 1.923
P = 0.1765
and phosphate performed poorly compared to roots grown on
medium supplemented with arsenic but performed better than
roots grown on medium containing only phosphate.
Given these results we repeated the experiment in entirety.
Medium and root stocks were newly prepared. Roots in the
second study were allowed to grow under conditions identical
to those of the first group except the cultures were maintained
for twenty-one days instead of eighteen. Results of the second
experiment almost identically matched the results of the first
study (Table 3).
ICP-MS was used to determine if any contaminants were
present in the arsenic standard solution that may contribute as
macro- or micronutrients. The results of the ICP-MS analysis
indicated that no unexpected elements were present in the
standard solution.
We have shown that, in the context of this study, low
concentrations of arsenic contribute to the development and
growth of root tissue. We do not yet understand the physiological or biochemical causes for this phenomenon. Many
studies exist where arsenic at higher levels (e.g. 100 mg L−1 )
are examined and have shown phytotoxicity. This study supports the conclusion that at low concentrations (100 µg L−1 )
arsenic is not phytotoxic to corn roots grown in culture. New
studies are underway which will help ascertain the level at
which arsenic becomes overtly phytotoxic. As a follow-on
study to the work presented here we are examining the ultrastructure of root tip mitochondria using transmission electron
microscopy.
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G. Evans et al.
(a)
(b)
(c)
(d )
Fig. 1. Photographs of corn roots (Z. mays) grown on media containing (a) 100 µg L−1 As and 0.0 mg L−1 KH2 PO4 , (b) 100 µg L−1 As and
4.1 mg L−1 KH2 PO4 , (c) 0.0 µg L−1 As and 4.1 mg L−1 KH2 PO4 , and (d) 0.0 µg L−1 As and 0.0 mg L−1 KH2 PO4 .
In spite of the large amount of recent research into the role
of arsenic in biological systems, there are aspects of arsenic
we do not understand. It may be that in some situations, and at
very low levels, arsenic may stimulate root growth, ultimately
leading to a selective advantage for plants that exhibit such
behaviour.
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Acknowledgment
We acknowledge the financial assistance of the National Science Foundation for grants DBI-0244221 and CHE-0116804,
and the USA Department of Energy, through cooperative
agreement no. DE-FC02–02-EW15254, administered by the
HBCU/MI Environmental Technology Consortium.
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