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The Effect of Nitrate on the Uptake of Pertechnetate in Wheat
By Jack Butt (08011208)
Supervisor: Dr Neil Willey
Project Title: The Effect of NO3- on the Uptake of 99TcO4- in Wheat.
Jack Butt 08011208
BSc (Hons) Environmental Biology Final Year 2011-2012
This submitted project is my work, it contains no
unreferenced or unacknowledged verbatim extracts from the
works of others and it has not (either in whole or in part)
been submitted towards the award of any other award either
at UWE or elsewhere.
Signed.................................................................................
Date...............
1
Acknowledgments
I would like to thank Dr Neil Willey, Alison Halliday, Rihannon Davis, David Molesworth, Marcus
Pugh and Steve Bride for their support and help throughout all of the aspects that went into writing
this report. I would also like to thank all of my friends and family for their continued support
throughout my project and all of my studies.
2
Table of Contents
Table of Figures.................................................................................................................................................4
1: Abstract.............................................................................................................................................................4
2: Introduction.......................................................................................................................................................5
3: Methodology......................................................................................................................................................6
3.1: Overview....................................................................................................................................................6
3.2: Growing the Wheat.....................................................................................................................................6
3.3: Experimental Procedure.............................................................................................................................7
3.4: Statistical Analysis....................................................................................................................................11
4: Results.............................................................................................................................................................12
4.1: Experiment 1 - The investigation into the effects of increasing nitrate concentrations on pertechnetate
uptake in nitrate and ammonium grown plants...............................................................................................12
4.2: Experiment 2 – The investigation into the uptake of pertechnetate in ammonium, nitrate and glycine
grown wheat plants treated with sulphate, phosphate and nitrate.................................................................13
4.3: Experiment 3 – The investigation into uptake of pertechnetate by glycine grown wheat plants treated
with increasing concentrations of nitrate, ammonium and glycine.................................................................17
5: Discussion........................................................................................................................................................18
5.1: Determining the mechanism of pertechnetate uptake.............................................................................18
5.2: The mechanism of pertechnetate uptake.................................................................................................20
5.3: Translocation of pertechnetate................................................................................................................21
5.4: Further investigation................................................................................................................................21
6: Conclusion.......................................................................................................................................................22
7: Appendices......................................................................................................................................................23
7.1: Appendix 1 Experiment 1 Statistics Output..............................................................................................23
7.2: Appendix 2 Experiment 2 Statistics Output..............................................................................................25
7.3: Appendix 3, Experiment 3 statistical output.............................................................................................27
8: References.......................................................................................................................................................30
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Table of Figures Figure 1:..............................................................................................................................................8
Figure 2:............................................................................................................................................10
Figure 3:............................................................................................................................................12
Figure 4:............................................................................................................................................13
Figure 5:............................................................................................................................................15
Figure 6.............................................................................................................................................16
Figure 7:............................................................................................................................................17
1: Abstract Technetium -99 (99Tc) is a low energy β- -emitting radioisotope. It has been suggested that leakages
from underground storage repositories as the most probable source of environmental
contamination with 99Tc. In aerobic soils 99Tc exists as pertechnetate, which presents a problem
because pertechnetate is mobile in soils and readily taken up by plants. When taken up by plants
it’s translocated to the leaves and transported up the food chain by anything that eats them.
Understanding how plants take up pertechnetate is of significant importance. Increasing nitrate
concentrations in soils decreases pertechnetate uptake, indicating common uptake through nitrate
channels, there is also a competition between sulphate, phosphate and molybdate. This report
aimed to evaluate nitrogen sources to ascertain whether pertechnetate is entering the plants
through the nitrate channels by competition experiments between nitrate and other mineral ions.
The investigation was carried out using hydroponically grown wheat, using different nitrogen
sources and treatments. The investigation found that when wheat plants, grown in an ammonium,
nitrate or glycine Hoaglands solution, were radio-labelled with pertechnetate and treated with
1.0mM of nitrate for 24hours, It led to the expression of high affinity NO3- channels and led to a
significant uptake of pertechnetate ions. Indicating, that uptake of pertechnetate happens via
nitrate channels. This report also found that ammonium, nitrate and glycine grown wheat plants
radio-labelled with pertechnetate and treated with 1.0mM of phosphate and sulphate showed
competition for uptake, indicating pertechnetate enters plants through sulphate and phosphate
channels also. The report also found treatment with either phosphate, sulphate or nitrate
significantly affects transfer of pertechnetate from roots to shoots.
4
2: Introduction 99Tc is a low energy β- -emitting radioisotope (Willey et al.2010) and has a 6% fission yield from 238U
and 239 Pu (Mousny et al, 1979), with a half-life = 2.13 x105 years (Cataldo et al, 1983). Experiments
carried out by Murphy and Johnson (1993) showed leaching from underground storage repositories
a potential channel for 99Tc contamination of soils. Salt-stone waste lysimeters set up by murphy
and Johnson at the Savannah River laboratory showed a significant migration of 99Tc from
lysimeters to soil and plants. Mousny et al (1979), and Bennett and Willey (2002) also argue that
the storage of nuclear waste in underground repositories presents a significant route for soil
contamination with 99Tc. When released into soil systems 99Tc can exist as pertechnetate (99Tc04),
which is very mobile within the soil and bio-accumulated by plants. 99Tc can also exist as 99Tc02+.
99Tc02+ is of less environmental concern because it is relatively immobile in the soil and not readily
bio-accumulated by plants. The existence of these two ions within the soil system depends
primarily on soil redox.
Under anaerobic reducing conditions 99Tc exists as 99Tc02+, Ishii et al (2007) found that formation
99Tc02+ is also linked to bacterial metabolism. Under oxidising conditions 99Tc exists as
pertechnetate (Bennett and Willey, 2002). 99Tc02+ is of less environmental concern, but still
presents a problem as a lot of soils alternate between oxidising aerobic conditions and reducing
anaerobic conditions overtime time, especially in areas with a changeable water table (Bennett and
Willey, 2002). Therefore 99Tc fixed in an anaerobic soil as 99Tc02+ has the potential to be re-oxidised
into pertechnetate and migrate upwards through the soil.
99Tc in soils is of environmental significance because many plant species bio-accumulate
pertechnetate ions. Cataldo et al. (1983) witnessed significant uptake of pertechnetate ion in soya
bean plants, Echevarria et al (1998) also witnessed a similar phenomenon with uptake of
pertechnetate by ryegrass and wheat. Willey et al (2010) presents bio-accumulation data recorded
from 116 different plant species and also reports a significant uptake of pertechnetate ions by
other food crops. Furthermore Mousny et al (1979) present data showing significant translocation
of 99Tc within pea plants, with a significant proportion of total accumulated 99Tc found in the leaves.
Bio-accumulation of pertechnetate ions is of significant importance to ecological and agricultural
systems because it’s a source of transport of 99Tc up food chains and contamination of food crops,
especially as many staple food crops readily accumulate 99Tc (Willey, et al, 2010).
Understanding how pertechnetate enters into plants is of significant importance for determining
stratigies for dealing with 99Tc environmental contamination. The aim of this report is to establish
5
the mechanism of pertechnetate ion uptake. Cataldo et al. (1983) argues, pertechnetate uptake is
metabolically mediated and also affected by certain mineral ions, furthermore Echevarria et al
(1998) showed a competition between application of nitrate and pertechnetate uptake by plants,
hinting towards an interaction between pertechnetate and nitrate transporters. More recent work
carried out by willey and bennett (2002) describes uptake of pertechnetate through nitrate
channels as a probable method of pertechnetate uptake. This report is going to determine whether
pertechnetate enters plants through nitrate channels by comparing;
Pertechnetate emissions in nitrate and ammonium grown wheat plants under increasing
nitrate concentrations.
Pertechnetate emissions in nitrate, ammonium and glycine grown wheat plants in the
presence of sulphate phosphate and nitrate.
Pertechnetate emissions in glycine grown wheat plants under increasing nitrate,
ammonium and glycine concentrations
3: Methodology 3.1: OverviewThree experiments were carried out to examine the effect of nitrate on uptake of Pertechnetate by
wheat plants. Experiment 1 was investigated the effect of increasing nitrate concentrations on the
uptake of pertechnetate by wheat plants grown using two different nitrogen sources. Experiment 2
investigated the effect of nitrate, phosphate and sulphate on pertechnetate uptake by wheat grown
using three different nitrogen sources. Experiment 3 looked at the effect of increasing nitrate,
ammonium and glycine concentrations on uptake of pertechnetate by wheat over time, with
glycine as a nitrogen source.
3.2: Growing the WheatThe wheat was grown hydroponically in a modified Hoaglands solution. Using vermiculite as the
substrate, 10-15 wheat seeds were planted in small cylindrical pots. In batches, these pots were
suspended in deionized water and left to germinate. Pots were suspended in a hydroponic tank,
split into four compartments. Each compartment had a capacity for 15 pots and could hold 40L of
solution. The hydroponic tank was aerated using a small electric pump. Following germination the
deionized water was replaced with a modified Hoaglands solution. The setup of the hydroponic
system is detailed in figure 1. The constituent mineral ions of the modified Hoagland solution were
made up in separate 1.0M solutions, with the exception of the micronutrients. These were
6
combined to make one 5x10-2mM micronutrient solution. The constituent mineral ions were added
directly to each compartment. Each compartment contained 40L of Hoaglands solution with the
following nutrients:
Calcium Chloride (CaCl2), 1.00x10-0mM.
Potassium Chloride (KCl), 2.50x10-1mM.
Magnesium Sulphate (MgSO4), 1.00x10-1mM.
Di-Sodium Hydrogen Phosphate (Na2HPO4), 4.00X10-1mM.
Iron EDTA (Fe EDTA), 1g/100ml
Di-Hydrogen Borate (H2BO3), 2.860g per L.
Zinc Sulphate (ZnSO4), 0.220g per L.
Copper Sulphate (CuSO4), 0.790g per L. Micronutrients
Manganese Sulphate (MnSO4), 1.015g per L.
Di-Hydrogen Molybdate (H2MoO4), 0.090g per L.
The above mineral ions remained constant in each of the modified Hoagland solutions. The nitrogen
sources differed between solutions; the following mineral ions were used as the sources of
nitrogen:
Calcium Nitrate (Ca(NO3)2), 1.00x10-0mM.
Ammonium Chloride (NH4Cl2), 2.00x10-0mM.
Glycine (NH2), 2.00x10-0mM.
7
3.3: Experimental ProcedureFigure 1 : A diagram outlining the specifics of the hydroponic system used in this investigation.
Rubber Seal
Vermiculite Substrate
Cylindrical Tube
Wire Mesh
8
Air Pump
Green House:
Maintained between 10 and 250C
Hydroponic
Tank
Hoaglands
Solution
Hydroponic pots
with wheat and
substrate, black
rubber seal
prevents pots from
falling into tank
Each sheet can hold 15 pots - 60
pots per tank.
Green House was lit with artificial
lighting between 6 and 9, am and
pm
Sheets in
which pots
are
suspended
The first round of experimental testing was carried out on 24 pots of wheat plants. Twelve pots
were grown with calcium nitrate as the source of nitrogen and 12 with ammonium chloride. Plants
were 7 weeks old when they were radiolabelled with technetium99. Radiolabelling was carried out in
the radiation labs at the university. The batch was split into two separate groups – ammonium
grown and nitrate grown. Each separate batch was then split into four groups of three pots; each
group was then subjected to four different treatments. The treatments were pots placed in a
solution containing pertechnetate and deionized water for 24 hours. Pots placed in a solution
containing pertechnetate and 0.1mM of calcium nitrate for 24 hours. Pots placed in a solution
containing 99Tc and 0.5mM of calcium nitrate for 24 hours. Pots placed in a solution containing
pertechnetate and 1.0mM of calcium nitrate for 24 hours The solutions used in each treatment
were made prior to experimental testing and radiolabelled with 40KBq/L of pertechnetate. Solutions
were then decanted into conical flask and pots placed in the top. Conical flasks were then placed in
a lighted incubator for 24 hours. The experimental set up is detailed in figure 2.
Uptake of radiation was determined by digesting the plant and using a beta counter to measure
pertechnetate emissions. After 24 hours plant shoots were harvested, weighed and placed into
envelopes to be dried in an oven. After at least 24 hours of drying the plant shoots were then re-
weighed. After total dry weight was taken, 0.1g of plant material was placed into a separate boiling
tube. To which 5mL of nitric acid was added and left for 12 hours at room temperature. After 12
hours the nitric acid was then heated to 800C for 1 hour, 5mL of hydrogen peroxide was then added
and kept at 800C and left until bubbling ceased. Once bubbling had ceased 0.1mL of the digested
plant material was added to 10mL of scintillant and placed into the beta counter to measure
pertechnetate emissions.
9
Figure 2 : A diagram outlining the genernal experimental setup of how each wheat plant will be treated in each experiment.
The second round of experiment testing was carried out on a batch of 27 pots. The plants were
grown on three different nitrogen sources. To start all 27 pots were grown on a nitrate based
Hoaglands solution. Five days prior to experimental testing the nitrogen sources for 18 pots were
changed. Nine were placed in a Hoaglands solution with ammonium chloride and nine with glycine.
The plants were three weeks and five days old when they were radiolabelled. The experimental
setup for experiment 2 was the same for experiment 1 and detailed in figure 2. The treatments
consisted of 3 pots from each of the three different groups of plants placed in a solution containing
1.0mM of calcium nitrate radiolabelled with pertechnetate for 25 hours. Another 3 pots from each
of the three different groups of plant placed in a solution containing 1.0mM of di-sodium hydrogen
phosphate radiolabelled with pertechnetate for 25 hours. 3 pots from each of the three different
groups of plants placed in a solution containing 1.0mM of magnesium sulphate radiolabelled with
pertechnetate for 25 hours. The three solutions were radiolabelled with 24KBq/L of pertechnetate
and decanted into the conical flasks. After 25 hours a 0.5mL sample of solution was taken from
each conical flask, placed in 5mL of scintillant and put into the beta counter to measure
10
Conical Flask: Can hold
between 350 and 400mL of
solution, depending on pot
size
Hydroponic pot with wheat plants: Sits
in top of conical flask, held in place by
rubber seal around pot.
Treatment Solution
pertechnetate emissions. The shoots were then harvested, dried, digested and measured for
pertechnetate emissions.
Experiment 3 was carried out on 27 pots of wheat plants all grown on a glycine Hoaglands solution.
In this experiment there were nine treatments. These were arranged as displayed in figure 2. The
treatments were pertechnetate and glycine in 1.0mM, 5mM and 10mM solutions. pertechnetate
and calcium nitrate in 1.0mM, 5.0mM and 10mM solutions.pertechnetate and ammonium chloride
in 1.0mM, 5.0mM and 10mM solutions. There were three replicates for each treatment and they
were left for 48 hours. Depletion was taken at 0, 1, 4, 6, 23, 25 and 48 hours, in method outlined in
experiment 2. The pertechnetate content for the solutions was 24KBq/L and the wheat was 4
weeks and five days old when it was irradiated
3.4: Statistical AnalysisData was recorded and entered in to Microsoft excel. The data was then subsequently transferred
into IBM SPSS statistics 19 for statistical analysis. Statistical analysis was carried out on shoot dry
weight (of the acid digest plants), on the radioactivity (Bq/g) and total absorbed radioactivity (Bq).
Statistical analysis was carried using two and one way ANOVAs, with post hoc test where
applicable. All statistical print outs are presented in the appendix.
11
4: Results 4.1: Experiment 1 - The investigation into the effects of increasing nitrate concentrations on pertechnetate uptake in nitrate and ammonium grown plantsFigure 3 : The average pertechnetate activity of shoots harvested from seven week old hydroponically grown wheat plants, grown using two different nitrogen sources(Ammonium and Nitrate), plants were radiolabelled with pertechnetate (40Bq/mL) and then treated with increasing nitrate concentrations (0.0mM, 0.1mM, 0.5mM and 1,0mM) over a 24 hour period.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10
100
200
300
400
500
600
700
800
Nitrate Grown Plants Ammonium Grown Plants
Nitrate Concentration (mM)
Radi
oacti
vity
(Bq
/g)
Statistical analysis of shoot weights revealed no significant difference between dry weight of nitrate
or ammonium grown plants (df 1, f = 0.021, p = 0,887). Univariate analysis of total pertechnetate
activity of plant shoots revealed no significant difference between treatment and nitrogen source
(appendix 1). However, analysis of pertechnetate activity per gram of dry weight did reveal
information about the effect of nitrate on pertechnetate uptake. Univariate analysis of shoot
activity revealed a relationship between nitrogen source and treatment (df 3, f = 6.359, p = >
0.01). Further analysis revealed no significant effect of treatment on pertechnetate activity in
nitrate grown plants (df 3, f = 3.137, p = 0.087). However, further analysis did reveal a significant
difference between treatment effect on radioactivity in ammonium grown plants (df 3, f = 6.506, p
= 0.015).
The above figure shows a decrease in radioactivity in ammonium plants treated with 0.1mM of
nitrate in comparison to the control plants, which are more radioactive. With no evidence of
12
unequal variances ( df 3, Levene stat 2.544, p = 0.129) a post hoc tukey revealed that
radioactivity in ammonium grown plants treated with 0.1mM of nitrate is significantly different to
ammonium grown plants treated with no nitrate and 1.0mM of nitrate, but is not significantly
different to ammonium grown plants treated with 0.5mM of nitrate. However, pertechnetate
activity in the ammonium grown plants treated with 0.5mM of nitrate was not significantly different
to plants treated with 1mM and 0mM nitrate either. The figure also shows no significant decrease in
pertechnetate activity in nitrate grown plants. The main points that can be taken away from
experiment 1 are; ammonium grown plants take up pertechnetate without the addition of nitrate,
increasing nitrate concentrations did not significantly reduce pertechnetate uptake in nitrate grown
plants, a nitrate concentration of 0.1mM can significantly decrease pertechnetate uptake in
ammonium grown plants, but a concentration of 1.0mM does not.
4.2: Experiment 2 – The investigation into the uptake of pertechnetate in ammonium, nitrate and glycine grown wheat plants treated with sulphate, phosphate and nitrateFigure 4 : The average pertechnetate depletion of three weeks and five day old hydroponically grown wheat plants, grown using three different nitrogen sources (Ammonium, Nitrate and Glycine), plants were radiolabelled with pertechnetate (24Bq/mL) and then treated with either 1.0mM of nitrate , phosphate and sulphate and left for 25 hours.
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Nitrate Grown Plants Glycine Grown Plants Ammonium Grown Plants
Tota
l Rad
iatio
n De
plet
ed (B
q)
In figure 4 there is a large number of overlapping error bars which shows little variability in
pertechnetate uptake between nitrogen sources in each treatment group. Univariate analysis of
13
total depletion of pertechnetate with nitrogen source as the fixed factor and treatment as the
random factor, showed no significant difference in the effect of nitrogen sources on pertechnetate
uptake (df 2, f = 0.982, p = 0.450). Univariate analysis did show a strong significant difference in
treatment effect on pertechnetate uptake (df 2, f = 1487.93, p = >0.01), which is very apparent in
figure 4 due to the comparatively greater uptake of pertechnetate by plants treated with 1.0mM
nitrate. Univariate analysis also revealed no relationship between nitrogen source and treatment
(df 4, f = 1.565, p = 0.226), this is also apparent in figure 4 as there is much uniformity in response
from plants grown in each nitrogen source when exposed to the same treatments.
Univariate analysis of the remaining solution activity also revealed no significant difference in
nitrogen source effect on pertechnetate uptake (df 2, f = 989, p = 0.448) and a significant different
in treatment effect on pertechnetate uptake (df 2, f = 1487.783, p =>0.01), With no significant
relationship between the effect of nitrogen source and treatment. Statistical analysis of total
depletion using an ANOVA showed a significant difference in the effect of treatment on
pertechnetate uptake between treatments (df 2, f = 2043.561, p = >0.01).After a homogeneity
provided no evidence of unequal variances (df 2, Levene stat = 3.233, p = 0.057), further analysis
using a post hoc tukey test was carried out. Using the results from the post hoc tukey (df 2, p =
>0.01). and figure 4 it was clear that plants treated nitrate took up a significantly more
pertechnetate than plants treated with either phosphate or sulphate The post hoc tukey also
showed that there was no significant difference in pertechnetate depletion between plants treated
with phosphate or sulphate (df 2, p = 0.580).
Statistical analysis of remaining solution activity showed the same significant differences as the
above, the statistical outputs for these statistical tests can be found in appendix.2.
14
Figure 5 : The average pertechnetate content within shoots harvested from three weeks and five day old hydroponically grown wheat plants, grown using three different nitrogen sources (Ammonium, Nitrate and Glycine), plants were radiolabelled with pertechnetate (24Bq/mL) and then treated with either 1.0mM of nitrate , phosphate and sulphate and left for 25 hours.
Treated with 1.0mM sulphate
Treated with 1.0mM phosphate
Treated with 1.0mM nitrate0
50
100
150
200
250
Nitrate Grown Plants Glycine Grown Plants Ammonium Grown Plants
Tota
l Rad
iatio
n in
Sho
ots (
Bq)
Statistical analysis of shoot weights from experiment 2 gave no evidence of significant difference in
weight between nitrate, glycine or ammonium grown plants (df 3, f = 1.364, p = 0.278). Univariate
analysis of total shoot radiation content (Bq per shoot) using source of nitrogen as the fixed factor
and treatment as the random factor, showed no significant difference in the effect of treatments (df
2, f = 0.832, p = 0.499) or nitrogen source (df 2, f = 4.716, p =0.089). The analysis did reveal a
relationship between nitrogen source and treatment (df 4, f = 4.110, p = 0.015). Further analysis
using ANOVA test revealed no significant difference between treatment effect on nitrate grown
plants (df 2, f = 1.987, p = 0.218) or ammonium grown plants (df 2, f =4.526, p = 0.063). Further
analysis did reveal a significant difference in treatment effect on glycine grown plants (df 2, f =
13.266, p = >0.01). A homogeneity of variances test provided no evidence of unequal variances (df
2, Levene stat = 2.212, p = 0.191) and the following post tukey revealed that shoots of glycine
grown plants which were treated with phosphate contained a significantly different amount of
pertechnetate compared to the shoots of glycine grown plants that were treated with either nitrate
(df 2, p = >0.01) or sulphate (df 2, f = p = 0.022).this can be seen clearly in figure 5. Furthermore,
statistical analysis also revealed shoots of glycine grown plants that had been treated with either
sulphate or nitrate did not contain a significantly different amount of pertechnetate (df 2, p =
0.524).
15
Figure 6 The average pertechnetate activity in shoots harvested from of three weeks and five day old hydroponically grown wheat plants, grown using three different nitrogen sources (Ammonium, Nitrate and Glycine), plants were radiolabelled with pertechnetate (24Bq/mL) and then treated with either 1.0mM of nitrate, phosphate and sulphate and left for 25 hours.
Treated with 1.0mM sulphate Treated with 1.0mM phosphate
Treated with 1.0mM nitrate0
50
100
150
200
250
300
350
400
450
Nitrate Grown Plants Glycine Grown Plants Ammonium Grown Plants
Radi
oacti
vity
(Bq/
g)
Univariate analysis of shoot radioactivity per gram of dry weight showed a significant difference
between treatments (df 2, f = 16.621, p = >0.01). Further investigation using an ANOVA with
homogeneity of variances (df 2, Levene stat = 3.202, p = 0.59) and a post hoc tukey showed plants
treated with nitrate contained a significantly different levels of radiation (df 2, p = >0.01), which is
shown in figure 6 that Statistical analysis also showed plants treated with either phosphate or
sulphate did not contain significantly different levels of radiation (df 2,p = 0.133).
Experiment 2 found that there was significantly more depletion of pertechnetate by plants treated
with 1.0mM nitrate compared to plants treated with 1.0mM of either phosphate or sulphate. It was
also found that depletion of pertechnetate by plants treated with 1.0mM phosphate or sulphate was
not significantly different. Analysis of shoot radiation content showed significant translocation of
pertechnetate from roots to shoots in glycine plants treated with phosphate. Analysis of shoot
activity showed plants treated with 1.0mM of nitrate had the lowest activity levels in their shoots,
despite depleting the greatest amount of pertechnetate.
4.3: Experiment 3 – The investigation into uptake of pertechnetate by glycine grown wheat plants treated with increasing concentrations of nitrate, ammonium and glycineFigure 7 : Showing the average pertechnetate activity of the remaining treatment solution overtime, measurements were taken at 0, 1, 4, 6, 23, 25 and 48hours. The experiment was carried
16
using hydroponically grown wheat (four weeks and five days old), grown using one nitrogen source (Glycine), plants were radiolabelled with pertechnetate (24Bq/mL) and then treated with either 1.0mM, 5.0mM, or 10mM of nitrate , ammonium and glycine and left for 48 hours.
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.0027.50
28.00
28.50
29.00
29.50
30.00
30.50
31.00
Time Hours
Radi
oacti
vity
(Bq/
g)
Univariate analysis of total depletion of pertechnetate in experiment three showed no significant
difference in the effect of treatments and concentrations on pertechnetate uptake (Appendix 6.3).
However, further investigation of solution activities presented information. Figure 7 shows the
average solution pertechnetate activity over 48 hours. After 1 hour there is a small decrease in
pertechnetate activity, which is followed by a swift increase between 1 and 4 hours. After this point
pertechnetate activity swiftly decreases for an hour, after which the decrease is more gradual. At
23 hours there is another swift increase in pertechnetate activity for 2 hours. After 25 hours there
is a gradual increase in pertechnetate concentration up to 48 hours. Statistical analysis of
pertechnetate activity overtime showed pertechnetate concentration at four hours is significantly
different to starting pertechnetate activity (df 7, f = 582.369, p = 0.032) . However, pertechnetate
activity after 1, 6, 23, 25 and hours was not significantly different to the starting pertechnetate
activity.
Analysis of pertechnetate activity after 48 hours revealed a significant relationship between
nitrogen treatment and treatment concentration (df 4, f = 2.950, p = 0.49).Statistical analysis
using a one way ANOVA with a post hoc Games-Howell test showed this relationship appeared
between plants treated with ammonium at different concentrations. The test showed a significant
17
difference in pertechnetate activity in plants treated with 1.0mM of ammonium. Analysis of data
shows there is a significant difference because there is less pertechnetate activity at 1.0m
The results from experiment 3 show no significant difference in total depletion of pertechnetate
after 48 hours. Analysis of average pertechnetate activity overtime showed a significant increase in
pertechnetate activity after four hours, and no significant difference from starting concentration at
1, 6, 23, 25 and 48 hours (appendix 3). Analysis of pertechnetate activity after 48 hours showed a
significant difference in pertechnetate activity in plants treated with 1.0mM of ammonium, which
seemed to be that it had less activity.
5: Discussion 5.1: Determining the mechanism of pertechnetate uptakeBennet and Willey (2002) describes the potential likely-hood of pertechnetate uptake through
nitrate channels, specifically the 2H+/NO3- co-transporters. Previous work carried out by Echevarria
et al (1998) found a decrease in pertechnetate uptake with increased application of nitrate. The
theory is that pertechnetate is analogous to nitrate and other macro nutrient ions (Bennet and
Willey, 2002) and competes with these nutrients for uptake (Cataldo et al., 1983; Echevarria et al.
1998). Investigation into the effects of increasing nitrate concentrations on pertechnetate uptake in
nitrate and ammonium grown plants showed no significant competition for uptake between nitrate
and pertechnetate over 24hours, up to concentrations of 1.0mM of nitrate. Interestingly,
ammonium grown wheat plants treated with 1.0mM of nitrate, showed significantly more
pertechnetate activity. In comparison to ammonium grown wheat plants treated with 0.1mM of
nitrate and the groups of nitrate grown plants treated with nitrate. Some high affinity NO3-
transporters can react rapidly to external nitrate concentrations, and are also expressed in the
presence of external nitrate Increasing the capacity for nitrate uptake in the wheat plants(Crawford
and Glass, 2002; Maathius, 2009). This increase in uptake capacity would explain the absence of
competition effects under these conditions, because there could be more available area for nitrate
and pertechnetate take up. Leading to the high pertechnetate uptake witnessed in the ammonium
grown plants treated with 1.0mM nitrate and the nitrate grown plants , giving evidence to support
the theory that pertechnetate enters plants through nitrate channels. However, the high amount of
pertechnetate found in the control ammonium grown plants presents evidence to the contrary,
hinting towards the involvement of another uptake route.
18
Investigation into uptake of pertechnetate in ammonium, nitrate and glycine grown wheat plants
treated with sulphate, phosphate and nitrate showed interesting results. Nitrate grown plants
treated with 1.0mM of nitrate showed significant depletion of pertechnetate ions. Similarly to the
investigation into the effect of increasing nitrate concentrations on nitrate and ammonium grown
wheat plants, the significant uptake of pertechnetate ions can be attributed to the expression of
high affinity NO3- channels, brought on by external nitrate concentrations (Crawford and Glass,
2002; Maathius, 2009). Cataldo et al (1983) witnessed a competition between pertechnetate,
sulphate and phosphate. This experiment also showed a competition between pertechnetate,
sulphate and phosphate uptake, in wheat plants grown in all nitrogen sources . Cataldo et al’s
results showed a greater competition between phosphate and pertechnetate in comparison to
sulphate and pertechnetate, 32% and 61% (% of the uptake in the control plants) respectfully.
However, in this investigation depletion of pertechnetate in phosphate and sulphate solutions were
not significantly different from each other. Sulphate and phosphate ions typically exist in the
environment as either SO42- or H2PO4
- and enters plants through H+/SO42- and H+/H2PO4
- co-
transporters in the roots (Maathius, 2009). These channels are also described as possible
alternative avenues for pertechnetate uptake (Bennett and Willey, 2002).The potential for
pertechnetate to enter plants through phosphate and sulphate channels also explains the
significant uptake of pertechnetate by the control ammonium grown plants in the previous
experiment. The results from this particular experimental investigation show a repeated interaction
between nitrate and pertechnetate uptake and gives evidence for uptake of pertechnetate via
sulphate and phosphate channels. Suggesting that the mechanism of pertechnetate uptake in
plants involves 2H+/NO3, H+/SO42- and H+/H2PO4 channels.
Investigation into uptake of pertechnetate by glycine grown wheat plants treated with increasing
concentrations of nitrate, ammonium and glycine, shed little light on the mechanism of
pertechnetate uptake. Cataldo et al (1983) gave evidence that pertechnetate uptake was
metabolically mediated, data collected from this experimental investigation also showed evidence
that pertechnetate uptake is not a passive process. Figure 7 shows pertechnetate activity overtime,
and shows a significant difference in solution activity at fours hours. At four hours there was a
significant increase in pertechnetate activity, this indicates a concentration of the treatment
solution. This could only come about because there was no uptake of pertechnetate and water in
the treatment solutions was being lost by evaporation and transpiration. This indicates
pertechnetate uptake is an active process because transpiration was occurring, but uptake was not.
Bennett and Willey (2002) also argue that pertechnetate uptake is a metabolically mediated
19
process. This is because the exudation of H+ ions by plant roots makes the root cortex very electro
negative, which draws in cationic mineral ions, but means anionic mineral ions have to be taken up
actively (Bennet and Willey, 2002). The lack of pertechnetate uptake in this experiment can be
attributed to the poor health of the wheat plants used in this experiment. Temperature and light
conditions in the greenhouse at this point were non optimal, but time constraints meant plants had
to be used regardless of their condition.
5.2: The mechanism of pertechnetate uptakeThe investigations into the uptake of pertechnetate in ammonium grown plants under increasing
nitrate concentrations, and uptake of pertechnetate in ammonium, nitrate and glycine grown wheat
plants treated with sulphate, phosphate and nitrate, provides significant evidence that
pertechnetate is entering plants via nitrate channels. These investigations also gave evidence that
pertechnetate is entering plants through phosphate and sulphate channels as well. The final
investigation into the uptake of pertechnetate by glycine grown wheat plants treated with
increasing concentrations of nitrate, ammonium and glycine provided further evidence that
pertechnetate uptake is metabolically mediated. Showing the overall mechanism of pertechnetate
uptake involves 2H+/NO3, H+/SO42- and H+/H2PO4 channels. These finding are of importance for
predicting how pertechnetate behaves when released into the environment and in formulating
strategies to deal with environmental contamination.
20
5.3: Translocation of pertechnetateMousny et al (1979) and Mousny and Myttenaere (1981) present data showing significant
translocation of pertechnetate within pea plants, with a significant proportion of total accumulated
pertechnetate found in the leaves. The investigation into uptake of pertechnetate in ammonium,
nitrate and glycine grown wheat plants treated with sulphate, phosphate and nitrate also showed
translocation of pertechnetate. However, there was a significant difference in the amount of
translocation between treatments. The shoots of wheat plants treated with nitrate had the least
amount of pertechnetate activity and the shoots of glycine grown wheat plants treated with
phosphate had the largest amount of total pertechnetate uptake. This is of importance because it
presents avenues for the contamination of food chains with pertechnetate ( Mousny et al, 1979;
Mousny and Myttenaere, 1981).The differences witnessed in pertechnetate translocation under
treatment regimes could be important for determining strategies to reduce pertechnetate
translocation if accidentally released into the environmental.
5.4: Further investigationCleveland and Liptzin (2007) present evidence of a possible existence of a Redfield ratio in
terrestrial ecosystems. Furthermore, work carried out by Gusewell (2004) and Fujita et al (2010)
presents evidence on the effect of nitrogen to phosphorous ratios on nutrition and uptake of
mineral ions in plants species. Gusewell (2004) also reports on the active regulation of internal N:P
ratios by plant species. Gusewell (2004) also argues that when plants are deficient in nitrogen,
uptake of nitrogen is increased and uptake of phosphorous is decreased, and that the reverse
happens when plants are deficient in phosphorous. It is possible that variances in a plants internal
N:P ratios could have a significant effect on pertechnetate uptake. It is possible that by placing a
plant in an environment deficient in nitrogen or phosphorous can cause deficiency responses
overtime, resulting in the expression of certain genes. Many mineral ion deficiency responses result
in the expression of genes leading to a higher proportion of transport channels in plasma
membranes, such as nitrate deficiency (Crawford and Glass, 1998). The relationship between
nitrogen and phosphorous in plants could help to explain some of this report’s findings, such as the
high amount of pertechnetate activity in the ammonium grown plants in the control treatments
during the 1st experiment and the high amount of pertechnetate uptake in plants treated with
nitrate during the 2nd experiment . The plants in these experiments may have been affected by a
change in the internal ratio of nitrogen to phosphorous. In these experiments nitrogen was limited
in the 1st experiment, but not in the 2nd.In the 1st experiment this may have led to a deficiency
21
response leading to the expression of additional nitrate or phosphate channels, increasing the
available area for pertechnetate uptake. In the 2nd experiment a lack of phosphate in solution may
have led to active attempts by the plants to take up more phosphorous in order re-balance their
internal N:P ratio. N:P ratios may be important for the determining the extent of pertechnetate
uptake as this report gives evidence that pertechnetate uptake relies on phosphate and nitrate
channels. This could have implications for predicting how pertechnetate acts in the environment
and investigation into the effects of alternating external N:P ratios could be important to
understanding the mechanism of pertechnetate uptake and warrants further investigation.
6: Conclusion This report concludes that nitrate does have a significant effect on pertechnetate uptake. However,
this report cannot conclusively prove that pertechnetate enters plants specifically and only through
nitrate channels. A comparison of the experimental data, hints towards a complex relationship
between nitrate, phosphate and sulphate, and that application of these ions has the potential to
significantly alter the way in which pertechnetate behaves within the plants and affects the amount
of pertechnetate plants take up. This has serious implications for the study of pertechnetate uptake
in plants and the main conclusion of this report is , there is significant evidence that pertechnetate
enters the plant through 2H+/NO3, H+/SO42- and H+/H2PO4 channels, and that there needs to be
further investigation into the effect of nitrate, phosphate and sulphate application on the
translocation of pertechnetate in plants, and further investigation into the effect of alternating
external N:P ratios on uptake of pertechnetate.
22
7: Appendices 7.1: Appendix 1 Experiment 1 Statistics Output
ANOVA testing for a significant difference in shoot weight
ANOVA
DryWt
Sum of Squares df Mean Square F Sig.
Between Groups .000 1 .000 .021 .887
Within Groups .161 22 .007 Total .161 23
Total Shoot Radiation Univariate Analysis
Tests of Between-Subjects Effects
Dependent Variable:TotBq
Source
Type III Sum of
Squares df Mean Square F Sig.
Intercept Hypothesis 160733.760 1 160733.760 48.266 .006
Error 9990.433 3 3330.144a NSource Hypothesis 3447.845 1 3447.845 .857 .423
Error 12064.592 3 4021.531b Treatment Hypothesis 9990.433 3 3330.144 .828 .560
Error 12064.592 3 4021.531b NSource * Treatment Hypothesis 12064.592 3 4021.531 2.917 .066
Error 22057.127 16 1378.570c
a. MS(Treatment)
b. MS(NSource * Treatment)
c. MS(Error)
Shoot Activity Univariate Analysis
ANOVA testing for a significant difference in treatment effect on Shoot Activity in nitrate
grown plants
23
ANOVA testing for a significant difference in treatment effect on Shoot Activity in
ammonium grown plants, with homogeneity of variances and post hoc tukey
24
7.2: Appendix 2 Experiment 2 Statistics Output> An ANVOVA testing for a significant difference between shoot dry weight.
Total Depletion Univariate Anlysis
25
ANOVA Testing for a significant difference in total depletion between treatments, with
homogeneity of variances and post hoc tukey.
Univariate analysis of the activity in remaining treatment solutions
ANOVA Testing for a significant difference in activity of remaining treatment solution, with
homogeneity of variances and post hoc tukey.
26
7.3: Appendix 3, Experiment 3 statistical output Univariate analysis of total pertechnetate depletion after 48 hours
27
An ANOVA testing for a significant difference between starting activity and activity after 1,
4, 6, 23, 25 and 48 hours
Univariate analysis of the activity in the remaining treatment solutions after 24hours
ANOVA Testing for a significant difference in activity of remaining glycine treatment
solution
ANOVA Testing for a significant difference in activity of remaining nitrate treatment solution
28
ANOVA Testing for a significant difference in activity of remaining treatment solution, with
homogeneity of variances and post hoc games howell.
29
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