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Thammakul, W. and Ritchie R.J. (2019) Manganese toxicity in yeast and plants. 13th Botanical Conference of Thailand (BCT13), Department of Biology, Faculty of Science, Srinakharinwirot University 114 Sukhumvit 23, Bangkok, Paper 04-13, p 17-34, 14 – 15 June 2019.

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Abstract and Figures

Manganese (Mn) is normally present in soils as the insoluble, harmless MnO 2. The toxic Mn 2+ cation is formed in acid soils but there is little consensus on the physiological basis of Mn toxicity in plants. Yeast (Saccharomyces cerevisiae) and vascular plants share similar membrane transport mechanisms and so yeast provides a convenient model system for studies of Mn-toxicity. Early effects upon Mn toxicity in yeast in the exponential growth phase over of 24 h was examined in culture tubes and for 6 days in the freshwater aquatic angiosperm Lemna minor. In petri dishes at various Mn concentrations (10, 30, 100, 300, 1000, 3000 mmol m-3). Yeast grew exponentially and growth was followed by measuring optic density. Growth of L. minor was followed using leaf count, chlorophyll a content and absorptance of the plants. Mn has toxic effects on the yeast cells (K i = 1.325 ± 0.254 mol m-3) and L. minor (K i = 1.154 ± 0.282 mol m-3). Mn 2+ toxicity was reversible in yeast by a chelation agent (EDTA), but not in the case of L. minor. Therefore, our results showed that Mn is toxic to Yeast and L. minor (greater than or equal to 1 and 0.100 mol m-3 , respectively) and inhibited growth at even higher concentrations (more than 3 and 1 mol m-3 , respectively). Chelation of Mn consistently did not reduce the toxicity of Mn in L. minor.
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THE 13 BOTANICAL CONFERENCE OF THAILAND
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ความเจริญงอกงามทางพฤกษศาสตร์ พัฒนาคน พัฒนาชาติ เพ่ือความย่ังยืน
14 - 15 มิถุนายน 2562 คณะวิทยาศาสตร์ มหาวิทยาลัยศรีนครินทรวิโรฒ
Proceeding in the 13th Botanical Conference of Thailand
Manganese Toxicity in Yeast (Saccharomyces cerevisiae) and an Aquatic
Angiosperm (Lemna minor)
Wannapisit Thammakul1* and Raymond J. Ritchie1
Abstract
Manganese (Mn) is normally present in soils as the insoluble, harmless MnO2. The toxic Mn2+ cation
is formed in acid soils but there is little consensus on the physiological basis of Mn toxicity in plants. Yeast
(Saccharomyces cerevisiae) and vascular plants share similar membrane transport mechanisms and so yeast
provides a convenient model system for studies of Mn-toxicity. Early effects upon Mn toxicity in yeast in the
exponential growth phase over of 24 h was examined in culture tubes and for 6 days in the freshwater
aquatic angiosperm Lemna minor. In petri dishes at various Mn concentrations (10, 30, 100, 300, 1000, 3000
mmol m-3). Yeast grew exponentially and growth was followed by measuring optic density. Growth of L. minor
was followed using leaf count, chlorophyll a content and absorptance of the plants. Mn has toxic effects on
the yeast cells (Ki = 1.325 ± 0.254 mol m-3) and L. minor (Ki = 1.154 ± 0.282 mol m-3). Mn2+ toxicity was
reversible in yeast by a chelation agent (EDTA), but not in the case of L. minor. Therefore, our results
showed that Mn is toxic to Yeast and L. minor (greater than or equal to 1 and 0.100 mol m-3, respectively)
and inhibited growth at even higher concentrations (more than 3 and 1 mol m-3, respectively). Chelation of Mn
consistently did not reduce the toxicity of Mn in L. minor.
Keywords: Saccharomyces cerevisiae, Lemna minor, Manganese, Mn-toxicity
1 Faculty of Technology and Environment, Prince of Sonkla University: Phuket campus
* Corresponding author, e-mail: greenreadpean@gmail.com
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Proceeding in the 13th Botanical Conference of Thailand
Introduction
Manganese (Mn) is one of the most abundant metals in soils (7–9,000 ppm of Mn with an average of
440 ppm [1]) where it occurs as oxides and hydroxides, and it cycles through its various oxidation states as a
consequence of the redox state of the soils and microbial activity. Mn is a common element but is usually
present in soils as the virtually insoluble MnO2 and so concentrations of Mn2+ in free solution are usually very
low (1 mmol m-3 or less) [24]. Mn is essential to iron and aluminium alloys, steel and stainless steel
production [5–7] and so is ubiquitous in the waste flows of industrialized countries. Mn dioxide has some
industrial uses for example it can be converted to permanganate, a useful laboratory reagent [8], and is used
to produce chlorine and hydrochloric acid [9] and also used as a catalyst.
Mn is an essential trace nutrient for photosynthetic oxygen evolution in chloroplasts in terrestrial
plants and algae. It is also an essential trace element in higher animals, in which it participates in the action
of many enzymes [10]. It is an essential component of two key enzymes: the oxygen-evolving complex of
photosystem II and superoxide dismutase and much more extensively Mn acts as a redox cofactor for many
enzymes [2–4, 11, 12]. Plants normally obtain the trace amounts of Mn they require by secreting chelating
compounds or by dissolving MnO2 by H+-extrusion. Plants use these two processes to mobilize carefully
regulated amounts of Mn. Increased acidity in soils causes mobilization of Mn and Mn so can reach toxic
levels particularly in waterlogged soils [2–4, 10]. The conditions that mobilize Mn are also responsible for the
mobilization of toxic Al3+ and so the toxic effects of acid soils are often a combination of Al and Mn-toxicity [3].
Acid soils limit crop production, particularly of cereals, in much of the tropics and subtropics of Australia and
Asia. The combination of acid soils and waterlogging exacerbates Mn toxicity in plants [10].
The manganeseoxidizing group of microbes is a phylogenetically diverse assemblage
characterized by the ability to catalyze the oxidation of divalent, soluble Mn2+ to insoluble manganese oxides
of the general formula MnOx (where x is some number between 1 and 2) the organisms include a diverse
array of bacteria, fungi, cyanobacteria, eukaryotic algae, and other eukaryotic microbes [13]. Accumulations of
Mnoxides are noticeable as a dark brownblack precipitate. The relative abundance of different forms on Mn
are a function of the redox potential and oxygen levels.
Mn is an important element for human health, essential for development, metabolism, and antioxidant
biochemistry. However, excessive exposure or intake may lead to a condition known as Manganism. Effects
of excessive Mn effects occur mainly in the respiratory tract and in the brain, manganism is a
neurodegenerative disorder that causes dopaminergic neuronal death and symptoms similar to Parkinson's
disease [1, 14]. Manganism is typically an industry–related disease. In plants, Mntoxicity symptoms include
burning of the leaf margins and tips or as reddishbrown spot across older leaves. Chronic toxicity increases
the severity of the symptoms [3, 15, 16].
The aim of the present study was to compare Mn-toxicity in yeast (which shares many ion transport
mechanisms with higher plants, [17, 18]) with Mn-toxicity in a higher plant [24, 10, 15, 16, 19, 20]. Much
more is known about Mn as an essential element than Mn-toxicity. The freshwater aquatic L. minor is an
angiosperm but has a very simple anatomy. Toxicity of polyvalent cations is often a function of chelation state
[15, 16, 21–25] and so a study was made of the effects of the chelation agent ethylenediaminetetraacetic acid
(EDTA) on Mn-toxicity in both yeast and L. minor.
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Proceeding in the 13th Botanical Conference of Thailand
Materials and Methods
Chemical toxicity experiments
This study used Mn in the form of manganese (II) sulfate monohydrate (MnSO4·H2O) and
ethylenediaminetetraacetic acid (EDTA) in the form of disodium ethylenediaminetetraacetic dihydrate
(C10H14N2Na2O8·2H2O) for studies of the effect of toxicity on yeast (Saccharomyces cerevisiae) and L. minor.
The MnSO4 and EDTA solutions were prepared as stock solutions of 2, 100, and 500 mol m-3.
Yeast culture condition and growth measurement
Culture–yeast: The yeast strain used was a Bakers yeast strain from Assoc. Prof. Raymond J.
Ritchie, Prince of Songkla University, Phuket Campus, Thailand. Yeast was grown as stock cultures in
Wickerhams chemically defined medium (Table 1) [26]. Experimental cultures were incubated for a day at a
range of different concentrations of Mn (10, 30, 100, 300, 1000 and 3000 mmol m-3) and 1 mol m-3 EDTA.
The trace element and vitamin contents of the medium were as described by Zonneveld (1986) [26].
Chelators such as ethylenediaminetetraacetic acid (EDTA), citric acid, glutamic and malic were not included in
the culture medium and the experimental media because they would be likely to interfere with Mn toxicity.
Chelation agents tend to bind strongly to cells, so can be difficult to remove from cells, and so can seriously
interfere with experimental results.
Growth measurement: Yeast was grown in 200 µL aliquots of Wickerhams medium (pH adjusted to
7.5) and incubated at 30 °C in 96-well plates on an orbital shaker set to medium mode. The cultures were
therefore grown under aerobic conditions in the present study. The 96-well titer plates were read with a
standard Microplate Reader (A&E UK AMR–100, UK) at 630 nm (A630). Growth curves were fitted to a logistic
growth model. The exponential growth constant (k)(h-1) was determined by least squares fitting (EXCEL
Solver) and its asymptotic error determined by matrix inversion [17].
Table 1 Modified Wickerhams chemically defined medium.
Compound
Concentration
Glucose
55.5 mol m-3
KH2PO4
57.792 mol m
-3
MgSO4·7H2O
16.224 mol m-3
(NH4)2SO4
60.544 mol m
-3
H3BO3
8.09 mmol m-3
CaCl2·2H2O
68 mmol m
-3
ZnSO4·7H2O
1.39 mmol m-3
CuSO4·5H2O
0.16 mmol m
-3
NaI
0.56 mmol m-3
FeCl3·4H2O
0.739 mmol m
-3
NaMoO4
0.826 mmol m-3
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Proceeding in the 13th Botanical Conference of Thailand
Lemna minor material culture condition and growth measurement
Culture-L. minor: L. minor plants were cultured by using 10% BG–11 medium [27] in plastic cups.
Experiments were started with a single plant or a few plants and the growth was measured for 3-7 days under
a temperature of 30 ± 2 ºC under 24 h light using cool-white fluorescence light as described for growing C.
vulgaris above. After setting up a starter culture, L. minor plants were separated in 30 mL 10% BG–11 variant
media in petri dishes for an experiment under the same growth conditions, pH 7.50 ± 0.05 for 7 days under
different concentrations of Mn (10, 30, 100, 300 and 1000 mmol m-3) and ± 10 mmol m-3 EDTA where the
effect of a chelator was to be measured. In the case of L. minor, EDTA by itself was found to be relatively
non-toxic but was found to very toxic in the presence of elevated levels of Mn. This was a very different result
to that found in the case of yeast and C. vulgaris in the present study
Growth measurements: L. minor growth was easily measured by counting leaf number. Numbers of
leaf were counted as a simple measure of the plant growth. Growth analysis is a widely used analytical tool
for characterizing plant growth. Of the parameters typically calculated, the most important is the relative
growth rate (RGR), defined as the parameter r in equation 1.
RGR = ln (W
)2 - ln (W
)1
t2 - t1
(1)
where W1 and W2 are plants leaves number at time t1 and t2.
In each of the L. minor growth experiment the leaves were counted, the leaf number on the first count date
and the leaf count on the second date were recorded. RGR was then calculated for each experimental
treatment, and the values were averaged for the overall experiment. Alternatively, where growth was being
followed over several days, a curve was fitted to the lntransformed plant leaf number through time and RGR
at a particular time is calculated as the slope of the curve. When applied to counts made at only two points in
time, the results are algebraically identical to the RGR estimator (Equation 1) [28].
Photosynthesis measurement: L. minor plants were filtered by using vacuum filtration onto glass fiber
membrane filters. The photosynthesis of the flattened sample was measured using a PAM machine and Walz
software (Waltz, Germany) as relative photosynthetic Electron Transport Rate (rETR) as described above for
algal disks. Absorptance of the leaves was measured using the blue–ray RAT meter [18, 29, 30] to calculate
ETR from the rETR measurements calculated by the Walz software.
PAM machines measure photosynthesis on a surface area basis (mol e- m-2 s-1). Photosynthesis
measurements expressed on a leaf surface area basis have some uses in plant science, particularly in plant
ecology, but as in the case of algae, it is conventional to standardize photosynthetic rates on a Chlorophyll a
basis. Chlorophyll a was estimated on a leaf surface area basis using optical density (OD) measurements on
solvent extracts using a 7:2 mixture of acetone and ethanol because pure ethanol was not found to be a
satisfactory extractant for L. minor (Ritchie, 2018). The leaf samples were put in 10 mL centrifuge tubes and 3
mL 7:2 acetone/ethanol was added. The samples were incubated in the refrigerator at -10 ºC for about 12 h
in the dark. After removal from the refrigerator, the samples were kept at room temperature in a dark box and
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Proceeding in the 13th Botanical Conference of Thailand
then vortexed before being centrifuged to clear the Chlorophyll extract solution. One mL of cleared
supernatant was used for chlorophyll a determination using the Shimadzu spectrophotometer at 850, 665 and
648 nm using the equations of Ritchie (2018) for estimating the quantity of chlorophyll a (Equation 2) in 7:2
acetone:ethanol solvent. Using the Chl a/leaf surface area relationship it was possible to recalculate ETR as
mol e- g-1 Chl a s-1.
Chl a (µg/mL) = 2.34435 x (A648 nm A850 nm) + 12.4552 x (A665 nm A850 nm) (2)
Oxidation state of Mn at different pH.
Solutions were made up by adding different concentrations of Mn (0.5, 1, 5, 10 mol m-3). The pH of
the solutions was adjusted with 1% HCl (acid) and 10% NaOH (alkaline). pH was measured using a Lutron
PH-230SD pH meter (Lutron Electronics, Coopersburg, PA, USA).
Statistics
All results presented in this paper are means ± 95% confidence limits. Significantly different results
were identified using simple t-tests and ANOVA using the Tukey test interval (TTI) to detect differences
between mean parameters at the p < 0.05 level. Snedecor and Cochran [31] was used as the standard
statistical reference text.
Results
Manganese is normally present in soils as the insoluble, harmless MnO2. The toxic form of
manganese (Mn2+) is formed in acid soils but there is little consensus on the physiological basis of Mn toxicity
in plants. It has been reported that Mn toxicity in vascular plants could be reversed by chelation agents such
as EDTA [24].
Determination of the effects of Mn upon growth in yeast
Growth of the yeast was measured by following the optical density (OD) at 630 nm in a time course
of 24 h. If the A630 was greater than 1.0, the cell sample was diluted and the density of the culture calculated
from the diluent. Optical density is usually only directly proportional to cell numbers up to an OD of about 1 or
1.5. Growth of a control culture was included in each experiment. Growth was then followed for at least 24 h.
In the example shown, the effect of Mn upon exponential growth of yeast was determined at pH 7.5.
Chelation agents such as EDTA are often reported to control metal toxicity [21–23, 32–34]. The growth curves
of yeast in the conditions containing Mn and Mn plus EDTA are shown in Fig. 1a and 2, respectively. The
results show that the conditions of 1 mol m-3 Mn (Fig. 1a) and 3 mol m-3 Mn + 1 mol m-3 EDTA (Fig. 1b), had
almost identical inhibitory effects upon growth. The condition of 3 mol m-3 Mn almost halted growth. Inhibitory
effects were noticeable within 8 h of exposure.
Logistic exponential growth constant could be calculated using non-linear least squares fitting
methods using the Mn data shown in Fig. 1. A logistic modelling curve was fitted which took the lag-phase
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Proceeding in the 13th Botanical Conference of Thailand
into account. In the experimental conditions containing added Mn, the growth constants (h-1) of control (blank),
10, 30, 100, 300, 1,000 and 3,000 mmol m-3 Mn were 0.1017 ± 0.0155, 0.1036 ± 0.0177, 0.0131 ± 0.0156,
0.1044 ± 0.01650, 0.0955 ± 0.0135, 0.0682 ± 0.0057 and 0.0179 ± 0.0060, respectively. In the condition
containing Mn and EDTA, the growth constants (h-1) of control, 10, 30, 100, 300, 1,000 and 3,000 mmol m-3
Mn were 0.1233 ± 0.0315, 0.1201 ± 0.0276, 0.1200 ± 0.0294, 0.1170 ± 0.0255, 0.1052 ± 0.0196, 0.0919 ±
0.0132 and 0.0311 ± 0.0068, respectively.
Fig. 2 shows the exponential constants determined in an experiment similar to that shown in Fig. 1
plotted against the concentration of Mn ions in the absence and presence of EDTA. The inhibition constant
(Ki) for Mn and Mn + 1 mol m-3 EDTA were determined using non-linear least squares fitting. A students t-
test showed that the results were significantly different. The Ki of the Mn growth inhibition was 1.325 ± 0.254
mmol m-3 (r = 0.9714, n = 42) and that of the Mn plus EDTA condition Ki was 1.718 ± 0.288 mol m-3 (r =
0.9715, n = 42).
Determination of the effects of Mn upon growth in Lemna minor
Fig. 3 shows growth of the L. minor was measured by following the count leaf number over time.
Growth of a control culture was included in each experiment. Growth was followed for at least 6 days. In the
examples shown, the effect of Mn in the absence and presence of EDTA upon exponential growth of leaf
number of L. minor was determined at pH 7.5 as seen in Fig. 3a and 3b, respectively. Fig. 3a shows that
since 100 mmol m-3 in Mn had some inhibitory effects upon growth but not statistically significant. Growth of
the L. minor in 300 mmol m-3 Mn was almost halved and 1 mol m-3 Mn inhibited nearly all growth. Inhibitory
effects were visually noticeable within 2 and 1 days of exposure, respectively.
Exponential growth constant could be calculated using non-linear least squares fitting methods using
the Mn data shown in Fig. 3. The growth constants (h-1) of the control (blank), 10, 30, 100, 300 and 1,000
mmol m-3 Mn were 0.219 ± 0.0320, 0.2263 ± 0.0342, 0.1948 ± 0.0386, 0.1756 ± 0.0273, 0.1984 ± 0.0328 and
0.1040 ± 0.0353, respectively. In the condition containing Mn and EDTA, the growth constants (h-1) of the
control, 10, 30, 100, 300 and 1,000 mmol m-3 Mn were 0.1632 ± 0.0395, 0.1459 ± 0.0424, 0.1731 ± 0.0282,
0.1696 ± 0.0508, 0.1505 ± 0.0620 and 0.0089 ± 0.0122, respectively. EDTA did not protect L. minor from Mn-
toxicity, unlike the observations on yeast above.
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Proceeding in the 13th Botanical Conference of Thailand
Fig. 1 The effects of Mn (a) and Mn + 1 mol m-3 EDTA (b) on the exponential growth of yeast over a time course of 24 h. Cells
were grown in modified Wickerhams medium at pH 7.5.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0246810 12 14 16 18 20 22 24
A630
Time (h)
Effect of Manganese on Growth of Yeast
Control
10 uM
30 uM
100 uM
300 uM
1000 uM
3000 uM
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0246810 12 14 16 18 20 22 24
A630
Time (h)
Effect of Manganese + EDTA on Growth of Yeast
Control
Mn 1
Mn 2
Mn 3
Mn 4
Mn 5
Mn 6
b
a
Control
10 mmol m-3 Mn
30 mmol m-3 Mn
100 mmol m-3 Mn
300 mmol m-3 Mn
1 mol m-3 Mn
3 mol m-3 Mn
Control
10 mmol m-3 Mn
30 mmol m-3 Mn
100 mmol m-3 Mn
300 mmol m-3 Mn
1 mol m-3 Mn
3 mol m-3 Mn
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Proceeding in the 13th Botanical Conference of Thailand
Fig. 2 Effect of Mn upon exponential growth of yeast in the absence and presence of EDTA at pH 7.5. Growth constants are
based on growth at 7 time points over 24 hours including hour (0). The inhibition constants (Ki) for Mn in the absence and
presence of EDTA were significantly different and so there was a significant difference in Mn-toxicity in the absence and
presence of EDTA. The Ki of the Mn growth inhibition was 1.325 ± 0.254 mmol m-3 (r = 0.9714, n = 42) and that of the Mn plus
EDTA condition Ki was 1.718 ± 0.288 mol m-3 (r = 0.9715, n = 42).
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
-0.5 00.5 11.5 22.5 3
Exponential Constant (k) (h-1)
Manganese Concentration (mol m-3)
Effect of Manganese ±EDTA Upon the Exponential Growth of Yeast
[Mn]
Hill Curve Fit to Manganese Data
Series5
[Mn] + EDTA
Hill Curve Fit to Manganese Data
Series6
0
Mn Growth Constant
Mn Inhibition Curve
Fit
Ki = 1.325 ± 0.254 mol m-3 (r = 0.9714, n = 42)
Mn
+ EDTA Growth Constant
Mn
+ EDTA Inhibition Curve Fit
Ki = 1.718 ± 0.288 mol m-3 (r = 0.9715, n = 42) + EDTA
Inhibition Curve Fit
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Proceeding in the 13th Botanical Conference of Thailand
Fig. 3 L. minor was grown in 10% BG-11 at pH 7.5. Exponential curves for the (a) control had r = 0.9670, n = 21 and the (b)
control had r = 0.9070, n = 21. The highest Mn concentration (1 mol m-3) coupled with the presence of EDTA was toxic.
Fig. 4 shows chlorophyll a content of L. minor measured as chlorophyll a content (µg) per unit
surface area (10-6 m2). Fig. 4a shows that the 1 mol m-3 Mn treatment had a chlorophyll a content half less
than the control condition. In Fig. 4b, the condition of 1 mol m-3 Mn + 10 mmol m-3 EDTA was found to be
toxic to the plant (L. minor died).
In the comparison of the toxicity of Mn experiments, growth constants were also determined, which
were based on growth after 6 days including day (0) (total 7 time points). Growth of a control culture was also
measured in each experiment.
Fig. 5 shows relative growth rate for L. minor. Fig. 5a shows that the plant growths in the conditions
containing 10 to 300 mmol m-3 Mn were not significantly different (ANOVA, ratio; chl a/Leaf SA). In the
example shown, the effect of Mn (Fig. 5a) and (Fig. 5b) Mn + 10 mmol m-3 EDTA upon relative growth rate of
0
2
4
6
8
10
12
14
16
18
20
22
0123456
Leaf number (n)
Time (Day)
Exponential Growth Curve with Manganese
Least Squares Fit Mn Control
Least Squares Fit Mn 3.25 µM
Least Squares Fit Mn 9.75 µM
Least Squares Fit Mn 32.5 µM
Least Squares Fit Mn 97.5 µM
Least Squares Fit Mn 325 µM
0
2
4
6
8
10
12
14
16
0123456
Leaves number (n)
Time (Day)
Exponential Growth Curve with Manganese + EDTA
Least Squares Fit Mn Control
Least Squares Fit Mn 10 µM
Least Squares Fit Mn 30 µM
Least Squares Fit Mn 100 µM
Least Squares Fit Mn 300 µM
Least Squares Fit Mn 1,000 µM
Least Squares Fit
Control
Least Squares Fit
3.25
mmol m-3
Least Squares Fit 9
.75
mmol m-3
Least Squares Fit 32 5
a
Least Squares Fit Control
Least Squares Fit 10 mmol m
-3
Least S
quares Fit 30 mmol m-3
Least Squares Fit
100 mmol m-3
Least Squares Fit 300 mmol m
-3
Least Squares Fit 1 mol m
-3
b
Least Squares Fit Control
Least Squares Fit
10 mmol m-3
Least Squares Fit
30 mmol m-3
Least Squares Fit
100 mmol m-3
Least Squares Fit
300 mmol m-3
Least Squares Fit 1 mol m
-3
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Proceeding in the 13th Botanical Conference of Thailand
L. minor was followed. Plants were grown in 10% BG-11 medium at pH 7.5. For Figs 5 a and b, relative
growth rates were calculated using RGR-equation 2 plotting over time using data shown in Fig. 3.
The relative growth rate (RGR) are shown in Fig. 5. In the condition containing Mn, the RGR (n d-1 n-
1) of the control (blank), 10, 30, 100, 300 and 1,000 mmol m-3 Mn were 0.3006 ± 0.0230, 0.3006 ± 0.0230,
0.2670 ± 0.0675, 0.2407 ± 0.0177, 0.2681 ± 0.0407, 0.1465 ± 0.0282, respectively. In the condition containing
Mn and EDTA, the RGR of the control, 10, 30, 100, 300 and 1,000 mmol m-3 Mn were 0.2005 ± 0.0688,
0.1716 ± 0.1135, 0.2193 ± 0.0346, 0.2061 ± 0.1125, 0.1891 ± 0.14610 and 0.0192 ± 0.0688, respectively.
Fig. 6 shows the exponential constants determined in an experiment similar to that shown in Fig. 3
plotted against the concentration of Mn ions in the absence and presence of EDTA. The inhibition constant
(Ki) for Mn and Mn + 10 mmol m-3 EDTA were determined using non-linear least squares fitting. A student’s t-
test showed that the results were significantly different. The Mn, Ki = 1.154 ± 0.282 mol m-3 (r = 0.9039, n =
36) and Mn + 10 mmol m-3 EDTA, Ki = 544 ± 279 mmol m-3 (r = 0.8650, n = 36).
Fig. 7 shows a rapid light curve for control L. minor plants. This light saturation curve is typical of
plants grown under low light conditions [18]. Photosynthetic ETR measured on a leaf surface area basis were
converted to a chlorophyll a basis (mol e- g-1 Chl a s-1) using measurements of Chl a per unit leaf surface
area. The optimum irradiance (EOpt) was about 300 µmol photon m-2 s-1 which is rather similar to the
conditions under which the plants were grown. The maximum photosynthetic electron transport rate is also
typical of plants grown under low-light conditions. Fig. 8 shows the maximum photosynthetic yield (Ymax) and
ETRmax for L. minor grown in a range of Mn concentration for 36 h with and without 10 mmol m-3 EDTA. The
very low EDTA concentration had to be used because of the toxicity of EDTA shown in Fig. 4, 5 & 6. The
range of Mn concentrations did not show a high degree of toxicity to photosynthesis for the incubation time
(36 h) used. EDTA did not protect L. minor from Mn and seemed to exacerbate Mn toxicity at higher Mn
concentration. This confirms the observations made in the growth experiments.
The maximum photosynthetic yield (Ymax) are shown in Fig. 8. In the condition containing Mn, the
Ymax of control (blank), 10, 30, 100, 300 and 1,000 mmol m-3 Mn were 0.5992 ± 0.0334, 0.5889 ± 0.0412,
0.5957 ± 0.0542, 0.5482 ± 0.0528, 0.5751 ± 0.0406 and 0.5688 ± 0.0430, respectively. In the condition
containing Mn and EDTA, the Ymax of the control (blank), 10, 30, 100, 300 and 1,000 mmol m-3 Mn were
0.5992 ± 0.0334, 0.5650 ± 0.0419, 0.5865 ± 0.0375, 0.5403 ± 0.0358, 0.5362 ± 0.0437 and 0.6795 ± 0.0478,
respectively.
The maximum photosynthetic ETR (ETRmax) are shown in Fig. 8. In the condition containing Mn, the
ETRmax (in µmol e- g-1 Chl a s-1) of the control (blank), 10, 30, 100, 300 and 1,000 mmol m-3 Mn were
211.7581 ± 10.6329, 273 ± 20.8, 290 ± 34.9, 324 ± 17.2, 332 ± 10.6 and 281 ± 18.2, respectively. In the
condition containing Mn and EDTA, the ETRmax of the control (blank), 10, 30, 100, 300 and 1,000 mmol m-3
Mn were 212 ± 10.6, 372 ± 23.1, 332 ± 15.1, 381 ± 25.8, 234 ± 25.5 and 135 ± 9.35, respectively.
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Proceeding in the 13th Botanical Conference of Thailand
Fig. 4 Comparison of the toxicity of Mn in experiments under the conditions containing Mn (a) and Mn plus EDTA (b). The
growth constants are based on growth at 6 days. The condition containing 1 mol m-3 Mn had chlorophyll a content half less than
the control plants. The condition containing 10 mmol m-3 EDTA and 1 mol m-3 Mn had no chlorophyll a content in the leaves
because the plants were killed. Chlorophyll a in each experiment was measured by using chlorophyll a content (µg) on a leaf
surface area (10-6 m2) basis.
0
2
4
6
8
10
12
00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Absorbance (Chl aµg)
Leaf SA (10-6 m2)
Lemna minor Chl aper leaf Surface Area in Different Manganese Concentration
Control Control Fit
3.25 µM 3.25 µM Fit
9.75 µM 9.75 µM Fit
32.5 µM 32.5 µM Fit
97.5 µM 97.5 µM Fit
325 µM 325 µM Fit
0
2
4
6
8
10
12
14
00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 11.1 1.2
Absorbance (Chl aµg)
Leaf SA (10-6 m2)
Lemna minor Chl aper leaf Surface Area in Different Manganese Concentration + EDTA
Control Control Fit
3.25 mmol 10 µM Fit
30 µM 9.75 mmol m-3 Fit
100 µM 100 µM Fit
300 µM 300 µM Fit
1,000 µM 1,000 µM Fit
Control
10 mmol m-3
30 mmol m-3
100 mmol m-3
300 mmol m-3
1 mol m
-3
Control
10 mmol m
-3
30 mmol m
-3
100 mmol m
-3
300 mmol m
-3
1 mol m
-3
a
Control Fit
10 mmol m
-3 Fit
30 mmol m
-3 Fit
100 mmol m
-3 Fit
300 mmol m
-3 Fit
1 mol m
-3
Fit
Control Fit
10 mmol m
-3 Fit
30 mmol m
-3 Fit
100 mmol m
-3 Fit
300 mmol m
-3 Fit
1 mol m
-3
Fit
b
27
Proceeding in the 13th Botanical Conference of Thailand
Fig. 5 Relative growth rate for L. minor in the conditions containing Mn (a) and Mn plus EDTA (b). Growth constants are based
on growth at 7 time points. Growth of a control culture was cultured in each experiment. Growth was then followed for 6 days.
Plants were grown in 10% BG-11 medium at pH 7.5.
0
0.1
0.2
0.3
0.4
0.5
0.6
0123456
Relative Growth Rate (RGR)
Time (day)
Lemna minor Relative Growth Rate vs. Time in Manganese Condition
Control
3.25 µM
9.75 µM
32.5 µM
97.5 µM
325 µM
0
0.1
0.2
0.3
0.4
0.5
0123456
Relative Growth Rate (RGR)
Time (day)
Lemna minor Relative Growth Rate vs. Time in Manganese + EDTA Condition
Control
10 µM
30 µM
100 µM
300 µM
1000 µM
Control
10 mmol m
-3
30 mmol m
-3
100 mmol m
-3
300 mmol m
-3
1 mol m
-3
Control
10 mmol m
-3
30 mmol m
-3
100 mmol m
-3
300 mmol m
-3
1 mol m
-3
b
a
28
Proceeding in the 13th Botanical Conference of Thailand
Fig. 6 Effect of Mn on the exponential growth of L. minor in the absence and presence of EDTA at pH 7.5. Growth constants
were based on growth at 6 days. The inhibition constants (Ki) for Mn in the absence and presence of EDTA were significantly
different so Ki could be calculated for Mn toxicity ±EDTA.
Fig. 9 shows the Mn oxidation state at different pH (7.5 and 4.5). Fig. 9 (a-d) shows the various
states (concentrations of 100, 2000, 5000, 10000 mmol m-3) of MnSO4 in pH 7.5. The solution had more
sediment when the Mn2+ was oxidized: the Mn2+ was transformed to Mn4+, forming insoluble MnO2 which is
non-toxic to L. minor. In contrast, Fig. 9 (e-h) shows the same concentration of MnSO4 in low pH. Sediment
was present in high Mn concentrations only. These results suggested that Mn2+ was non-oxidized and the
solutions were toxic to L. minor plants. However, this experiment is a preliminary test and needs to be
followed up. It might explain why chelation did not reduce Mn toxicity in L. minor.
Conclusion and Discussion
Manganese toxicity in yeast appears to be the same syndrome as Al-toxicity [32]. In yeast, Mn
toxicity can be conveniently measured by the effects of these metals on growth. The Ki of Mn was found to be
about 100 mmol m-3; toxicities of the metal could be reversed by EDTA. It is thought in vascular plants that
only the Mn2+ form is toxic [3]. However, toxicity in plants is claimed to be reversible by chelation by organic
acids such as malic, glutamic, oxalic and citric acids. Whether the observations that Mn toxicity is reversed by
the presence of different types of chelation agents such as citric acid in agreement with previous findings by
MacDiarmid and Gardner [33] or not has not been firmly established in this study in yeast. Citric acid is a
naturally occurring chelation agent that is secreted by roots. Citrate as a chelation agent in yeast needs
further study because it might have a much lower toxicity.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
-0.1 00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Exponential Growth Constant (k) (h-1)
Manganese concentration (mol m-3)
Effect of Manganese ±EDTA Upon the Exponential Growth of Lemna minor
Mn Growth Constant
Mn Inhibition Curve Fit
Mn + EDTA Growth Constant
Mn +EDTA Inhibition Curve Fit
0
Mn Growth Constant
Mn Inhibition Curve Fit
Ki 1.154 ± 0.282 mol m-3 (r = 0.9039, n = 36)
Mn
+ EDTA Growth Constant
Mn
+ EDTA Inhibition Curve Fit
Ki = 544 ± 279 mmol m
-3
(r = 0.8650, n = 36)
29
Proceeding in the 13th Botanical Conference of Thailand
Fig. 7 Photosynthesis of L. minor measured using PAM methods (n = 6). The Yield decays exponentially with increased
irradiance (Ymax = 0.5992 ± 0.0334, ½ Ymax at 107 ± 13.1 µmol photon m-2 s-1; Optimum irradiance = 287 ± 21.3 µmol photon m-
2 s-1 and ETRmax = 212 ± 10.6 µmol e- g-1 Chl a s-1.
Fig. 8 Photosynthesis of L. minor measured using PAM methods under a range of Mn concentrations ± EDTA (10 mmol m-3).
Plants were incubated for 36h. The Ymax without EDTA was almost independent of Mn concentrations. EDTA increased Ymax at
the highest Mn concentration. ETRmax tended to increase as Mn concentrations increased without EDTA and was inhibitory only
at the highest concentration of Mn. Mn + EDTA inhibited ETRmax at 300 and 1000 mmol m-3 Mn.
0
50
100
150
200
250
300
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0100 200 300 400 500 600 700 800 900 1000
PPFD Irradiance (µmol photon m2s-1)
ETR (µmol e-g-1 Chl a s-1)
Yield (Y)
Yield & ETR on Lemna minor Control
Yield- Control
Yield Fit - Control
ETR - Control
ETR Fit - Control
0
100
200
300
400
500
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
010 30 100 300 1000
ETRmax
Yieldmax
Manganese concentration (mmol m-3)
Mn Toxicity ±EDTA on Oxygenic Photosynthesis of Lemna minor (36 h)
Max Yield Mn
Max Yield Mn + EDTA
ETR Mn
ETR Mn + EDTA
30
Proceeding in the 13th Botanical Conference of Thailand
Fig. 9 Mn oxidation states (concentrations of 100, 2000, 5000, 10000 mmol m-3) of MnSO4 solution at different pH (7.5 and 4.5).
a-d are pH 7.5 and e-h are low pH.
Mn-resistant varieties of ryegrass (Populus cathayana) are known to secrete carboxylates [19, 21, 22,
34]. In the present study, attempts were also made to demonstrate reversal of Mn-toxicity in yeast and plant
using EDTA [10, 23, 35]. Preliminary experiments showed that EDTA was highly toxic (1 mol m-3) to yeast.
We found that yeast grew equally well on EDTA acid as with asparagine (Table 1). We found that EDTA-
grown yeast was found to be insensitive to Mn when tested in a modified Wickerhams medium without any
extra added vitamin (Table 1). Hence, EDTA will reverse the Mn-toxicity syndrome in yeast provided it is not
used at toxic concentrations (no more than 1 mol m-3 EDTA). Fig. 1a and 2 show that the effect of chelation
alleviated Mn toxicity uptake in yeast. Mn toxicity in yeast is not a simple function of the abundance of the
divalent forms of metal ions in the bulk medium. However, Fig. 1 shows that the initial OD of yeast in 3 mol
m-3 Mn were much higher than the other conditions because when 3 mol m-3 Mn was used and the pH
adjusted to 7.5, it had more sediment when the Mn2+ was oxidized to Mn4+ (insoluble MnO2) [10, 36]. Addition
of non-toxic levels of EDTA (1 mol m-3 EDTA) did not prevent the precipitation of MnO2.
It is extremely improbable that Mn2+ enters cells simply through the lipid bilayer. However,
interference with membrane function is thought to be a major factor in the toxicity of Mn2+ in addition to its
biochemical effects on enzyme function if it is in excess in the cytoplasm. Mn2+, like many other polyvalent
cations, is a potent channel-blocking agent due to the effects of Mn on calcium metabolism. Calcium is
practically universally involved in cell signaling and motility in plants and animals [2]. Any interference with this
function is likely to be toxic to cells. Mn is known to interfere with calcium function in vascular plants [4].
The low rates of uptake of Mn into the cytoplasm of cells is not a water–tight argument against the
idea that much of the toxicity of Mn is due to Mn that actually enters the cytoplasm of cells. Transport of Mn
ions within the xylem of land plants is essentially driven by mass upward flow of water created by the
transpiration stream and transport in the phloem is thought to occur via the positive hydrostatic pressure
gradient developed from the loading of sucrose into the phloem from mature actively photosynthesizing leaves
and unloading of sucrose into the sink tissues such as rapidly growing tissues, apical root zones and
reproductive organs [10, 20, 37–39]. L. minor is a small freshwater aquatic, but it still has roots and so there
is a transportation stream from the roots to the leaves and then out through the stomata by transpiration. The
a
b
c
d
e
f
g
h
31
Proceeding in the 13th Botanical Conference of Thailand
transpiration rate of water has a large effect on macronutrient translocation rate, however at low supply,
processes such as xylem loading and unloading and transfer between xylem and phloem have been shown to
be more important for the rate of nutrient supply [10, 39]. Mn in the xylem fluid exists in the uncompleted,
freeion form. Thus, in computer speciation studies, 37% of the Mn in Glycine max (Soybean) xylem sap and
72% of Mn in L. esculentum (tomato) xylem sap was found as Mn2+. Manganese was also found complexed
to organic acids (such as citrate) rather than by amino acids [24]. In another study, Mn in the xylem exudate
of Helianthus annuus (sunflower) was mainly present as Mn2+ at ranges of Mn supply from deficient to toxic
[10, 25]. Hence, the opportunity exists for high concentrations of harmful Mn2+ in the xylem fluid at excess Mn
supply. Mn is predominately transported throughout the plant within the xylem rather than the phloem [10, 40].
In another study, The metal toxicity may have an effect on the rate of xylem transport, possibly by reducing
transpiration [10, 41, 42]. This could have effects on the concentrations of other nutrients reaching the shoots.
As in the xylem, the pH, redox potential, ionic strength and organic constituents of the phloem sap will
determine the loading, transport and unloading of metals in the phloem. However, unlike xylem cells, phloem
cells are alive and metabolically active. Hence, metabolic reactions within the phloem have the potential to
make the phloem sap more responsive to changes in the internal plant environment than the xylem sap [10,
39]. Manganese mobility within the phloem is generally considered to variable and is dependent on the Mn
status of the plant species as well as the source and sink organs. Excess Mn supply reduced phloem
transport of Mn and the authors suggested loading from the xylem into the phloem may have been the rate
limiting process [10, 40]. L. minor has a limited growth of roots and probably much nutrient uptake occurs on
the underside of the floating leaves however, the leaves do have stomata and so there is significant
transpiration effects in L. minor just as in nonaquatic vascular plants.
We have shown that the presence of EDTA will protect yeast from Mn2+ toxicity. This is consistent
with another study which was on Al-toxicity in yeast [32], and studies on higher plants where chelation agents
are reported to have had the effect of reducing Mn toxicity in plants [19, 21, 22, 34]. In the present study, we
found that EDTA was ineffective in reducing toxicity in the aquatic L. minor and was highly toxic to L. minor
and had to be used at very low concentrations (Fig.s 4 to 8). In soil, Mn is present as manganese (II)
bicarbonate (Mn(HCO3)2), and soluble Mn2+ and Mn3+. When it occurs as oxides and hydroxides, it is
transformed to Mn4+ if oxygen is present into insoluble MnO2. Mn4+ is present as a dark brown-sediment
(MnO2) which is insoluble [2, 4, 10, 36]. EDTA has the effect of stabilizing solutions to prevent catalytic
oxidative decoloration, which is catalyzed by metal ions. The solubilization of Mn2+ ions can be accomplished
using EDTA [10, 43, 44]. That might explain why EDTA reduced Mn-toxicity in yeast condition (anaerobic) and
but could not reduce Mn-toxicity in L. minor (under aerobic conditions, grown under aquatic conditions)
because L. minor tolerates only very low concentrations of EDTA that were too low for effective chelation but
not EDTA toxicity.
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Lea's Chemistry of Cement and Concrete deals with the chemical and physical properties of cements and concretes and their relation to the practical problems that arise in manufacture and use. As such it is addressed not only to the chemist and those concerned with the science and technology of silicate materials, but also to those interested in the use of concrete in building and civil engineering construction. Much attention is given to the suitability of materials, to the conditions under which concrete can excel and those where it may deteriorate and to the precautionary or remedial measures that can be adopted.First published in 1935, this is the fourth edition and the first to appear since the death of Sir Frederick Lea, the original author. Over the life of the first three editions, this book has become the authority on its subject. The fourth edition is edited by Professor Peter C. Hewlett, Director of the British Board of Agrement and visiting Industrial Professor in the Department of Civil Engineering at the University of Dundee. Professor Hewlett has brought together a distinguished body of international contributors to produce an edition which is a worthy successor to the previous editions.
Article
The composition of xylem sap and exudate from stem incisions of Nicotiana glauca Grah. was compared in detail. Exudation from stem incisions occurred over a 5 min period in certain plants, enabling collection of 5–30 μl of sap. The rate of exudation showed an exponential decline. Exudate had a high dry matter content (170–196 mg ml⁻¹) and high sugar (sucrose) levels. Xylem sap had a low pH (5.8) and exudate a pH of 7.9. Glutamine dominated the amino compounds in xylem sap and exudate, and K⁺ was the major cation. Total amino compounds in stem exudate reached 10.8 mg ml⁻¹ whereas xylem sap contained much lower levels (0.28 mg ml⁻¹). All mineral elements and amino compounds with the exception of calcium were more concentrated in stem exudate than in xylem sap. Sucrose was labelled heavily in stem exudate following pulsing of an adjacent leaf with ¹⁴CO2. A concentration gradient of sugar (2.1 bar m⁻¹) was recorded for stems. Levels of sucrose, amino compounds and K⁺ ions in stem exudate showed a diurnal periodicity. Each commodity reached maximum concentration at or near noon and minimum concentration about dawn. The evidence suggests that exudate from stem incisions of N. glauca is a representative sample of solutes translocated in the phloem.