Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots
Abstract
Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.
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Blackwell Publishing LtdOxford, UKNPHNew Phytolo gist0028-646X© The Authors (2007). Journal compilation © New Phytologist (2007)234010.1111/j. 1469-8137.2007.02340.xDecember 200700899???906???Original Article sXX XX
Glyphosate inhibition of ferric reductase activity
in iron deficient sunflower roots
Levent Ozturk
1
, Atilla Yazici
1
, Selim Eker
2
, Ozgur Gokmen
1
, Volker Römheld
3
and Ismail Cakmak
1
1
Faculty of Engineering and Natural Sciences, Sabanci University, TUR–34956 Istanbul, Turkey;
2
Department of Soil Science, Faculty of Agriculture,
Cukurova University, TUR–01330 Adana, Turkey;
3
Institute of Plant Nutrition, University of Hohenheim, D–70593 Stuttgart, Germany
Summary
• Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent
glyphosate applications. A likely reason for this is that glyphosate interferes with root
uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot
and nongrass species.
• This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 m
M
glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose,
respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown
under Fe deficiency conditions.
• Application of 1.89 m
M glyphosate resulted in almost 50% inhibition of ferric
reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower
rates of glyphosate (e.g. 0.32 m
M and 0.95 mM), ferric reductase was inhibited.
Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots
were not decreased by the glyphosate applications.
• To our knowledge, this is the first study reporting the effects of glyphosate on ferric
reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase
could not be identified. Impaired ferric reductase could be a major reason for the
increasingly observed Fe deficiency in cropping systems associated with widespread
glyphosate usage.
Key words: chlorosis, ferric reductase, glyphosate, iron deficiency, sunflower
(Helianthus annuus).
New Phytologist (2008) 177: 899–906
© The Authors (2007). Journal compilation © New Phytologist (2007)
doi: 10.1111/j.1469-8137.2007.02340.x
Author for correspondence:
Ismail Cakmak
Tel: +90 216 4839524
Fax: +90 216 4839550
Email: cakmak@sabanciuniv.edu
Received: 14 September 2007
Accepted: 12 November 2007
Introduction
Iron (Fe) deficiency chlorosis is an important micronutrient
deficiency problem in crop production because it diminishes
growth, yield and nutritional quality of crops resulting in
serious economic and health implications. Large investments
are currently being made to correct Fe deficiency chlorosis. It
is estimated that the annual cost of overcoming Fe deficiency
is as high as 80–100
M Euros in the Mediterranean region
(Abadia et al., 2004) and 120
M USD in the north central
soybean-growing area of the USA (Hansen et al., 2004). Iron
deficiency also represents an important nutritional and health
concern in animal and human nutrition, resulting in severe
health complications in people, particularly in developing
countries (Welch & Graham, 2004). A major reason for the
widespread occurrence of Fe deficiency in humans is attributed
to the consumption of food crops containing low levels of Fe
as a result of various soil and genetic factors.
Iron deficiency in plants commonly occurs in soils that pro-
vide only low chemical and spatial Fe availability to plant roots.
Soil, climatic and plant factors have been reported to reduce
the availability and root uptake of Fe. Poorly aerated, cool
and calcareous or alkaline soils are characteristic soils where
Fe deficiency often occurs in plants (Marschner et al., 1986;
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Research
900
Inskeep & Bloom, 1987). Plants grown in such soils have
developed several adaptive mechanisms to improve Fe avail-
ability in the rhizosphere and to maintain sufficient root uptake
of Fe. These adaptive root responses to Fe-deficient soil condi-
tions differ among plant species. Dicots and nongrass monocot
species (Strategy-I plants) induce distinct root morphological
and physiological responses to Fe deficiency (Römheld, 1987;
Marschner & Römheld, 1994; Schmidt, 1999). An increased
capacity of roots to reduce Fe-chelates by an inducible ferric
reductase enzyme at the plasma membrane is the most charac-
teristic response of Strategy-I plants to Fe deficiency. Reduction
of Fe(III) to Fe(II) at the root surfaces is an obligatory process
in Fe acquisition of Strategy-I plants (Chaney et al., 1972;
Römheld & Marschner, 1986; Robinson et al., 1999).
Acidification of the rhizosphere by a proton-pumping ATPase
is another characteristic root response of Strategy-I plants to
Fe deficiency and contributes to increased solubility of Fe in
the rhizosphere (Marschner et al., 1986). Any impairment or
alteration in activity of these root mechanisms consequently
impairs Fe nutrition and induces leaf chlorosis, as shown in
plants grown in high bicarbonate soils (Alcantara et al., 2000),
or subjected to low temperature (Schmidt & Steinbach, 2000),
heavy metals (Barton et al., 2000) or ethylene inhibitors (Lucena
et al., 2006).
Iron deficiency chlorosis is also becoming increasingly preva-
lent in cropping systems receiving frequent or prolonged applica-
tions of glyphosate (N-phosphonomethyl glycine). Glyphosate
is the most extensively used herbicide in the world. Its usage
is increasing with the widespread cultivation of glyphosate-
resistant transgenic crops and the adoption of no-tillage crop-
ping systems (Cerdeira & Duke, 2006). This increasing usage
of glyphosate represents a potential threat to nontarget plants
because of glyphosate drift and accumulation in soils. Up to
10% of the applied rate of glyphosate can drift off target and
result in damage to nontarget plants, such as depressing nitro-
gen (N)-fixing capacity and disturbing nitrate assimilation
(Koger et al., 2005; Bellaloui et al. 2006; Buehring et al.,
2007). Foliar applied glyphosate to target plants is also effec-
tive in depressing root uptake of manganese of the nontarget
plants, indicating glyphosate transfer from target to nontarget
plants via the rhizosphere (Neumann et al., 2006).
Impairment of Fe nutrition in plants seems to be an adverse
effect of frequent glyphosate applications. Field observations
in many parts of the USA show that widespread occurrence
and increased severity of Fe deficiency chlorosis is associated
with frequent applications of glyphosate. Application of Fe
fertilizers has been shown to reduce the severity of chlorosis
and improve grain yield (Franzen et al., 2003; Hansen et al.,
2004; Jolley et al., 2004). These observations are in accord
with the recent findings that applying 1.89 mm glyphosate
(6% of the recommended dose) significantly decreases root
uptake and almost completely inhibits root-to-shoot trans-
port of Fe in sunflower plants within 12 h after glyphosate
treatment (Eker et al., 2006).
Based on these results and field observations, it may be
hypothesized that glyphosate restricts root uptake of Fe, by
inhibiting the development of root adaptation mechanisms
such as the root-cell ferric reductase activity. The objective of
the present study was therefore to investigate the effect of
glyphosate on root ferric reductase activity of sunflower plants
grown under Fe deficiency conditions. By inhibiting the activity
of the enolpyruvyl-shikimate-3-phosphate synthase (EPSPS),
glyphosate causes accumulation of shikimate in plants (Shaner
et al., 2005; Cerdeira & Duke, 2006). Shikimate is, therefore,
an important physiological indicator for the glyphosate activity
and glyphosate-related damage in plants (Mueller et al., 2003;
Shaner et al., 2005; Neumann et al., 2006). To determine the
degree of the glyphosate accumulation in roots after foliar
application, shikimate concentrations of roots were measured.
Since glyphosate application to leaves decreases sucrose export
into the sink organs (e.g. roots) (Geiger et al., 1999), and a
possible sugar shortage in the roots may limit ferric reductase
activity (Schmidt, 1999), the effect of glyphosate on the sugar
concentration of roots was also studied. To our knowledge,
this is the first study reporting the effects of glyphosate on ferric
reductase enzyme under Fe deficiency.
Materials and Methods
Plant growth
Sunflower (Helianthus annuus L. cv. TR-3080) plants were grown
hydroponically in a glasshouse equipped with an evaporative
cooling system (23–26 : 20–22°C day/night) under natural
daylight during the summer season in 2007 (location:
0°53′24.5″ N, 029°22′46.7″ E). Seeds were germinated in
Perlite moistened with saturated CaSO
4
solution for 5 d and
five uniformly selected seedlings were then transplanted to
plastic nutrient solution culture pots containing 2.7 l of
continuously aerated nutrient solution. The nutrient solution
(pH 5.5, unbuffered) contained: 0.75 mm K
2
SO
4
, 2 mm
Ca(NO
3
)
2
, 1 mm MgSO
4
, 0.25 mm KH
2
PO
4
, 0.1 mm KCl,
1µm MnSO
4
, 1 µm ZnSO
4
, 10 µm H
3
BO
3
, 0.1 µm CuSO
4
,
and 0.01 µm (NH
4
)
6
Mo
7
O
24
. Initially, Fe-deficient plants
were supplied with 0.2 µm Fe-ethylenediaminetetraacetic acid
(EDTA) for 5 d to avoid development of severe Fe-deficiency
stress. The concentration of Fe for Fe-sufficient plants was
100 µm and applied as Fe-EDTA. Six days after transferring
seedlings to the nutrient solution, Fe was withheld from the
solution of Fe-deficient plants for 5 d. After 11 d of growth in
nutrient solution under Fe deficiency, leaves started to show
slight chlorosis. With the onset of leaf chlorosis, glyphosate
treatments were applied as described below.
Foliar glyphosate applications
In all foliar applications, glyphosate formulated as Roundup Ultra
(active ingredient (ai): 480 g l
−1
N-[phosphonomethyl]glycine
© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 899–906
Research 901
isopropylamine salt; Monsanto Ltd, Adana, Turkey) was used.
The sublethal doses used for simulating foliar glyphosate drift
were 1, 3, and 6% of the recommended application rate (for
narrow- or broad-leaf annual weeds), as shown on the product
label (i.e. 1.44 kg ha
−1
ai glyphosate applied with 200 l of
water ha
−1
: 31.55 mm glyphosate as active ingredient), which is
equivalent to 0.32, 0.95, and 1.89 mm glyphosate, respectively.
Glyphosate solutions were freshly prepared before foliar
treatment. Application was made to the leaves using a plastic
hand-sprayer. Glyphosate was sprayed until all leaves became
wet (nearly 1.5 ml per plant), but without any run-off.
Root ferric reductase activity
To quantify ferric reductase activity of roots, intact plants were
incubated in 5 mm 2-(N-morpholino) ethanesulfonic acid
(MES) buffer (pH 5.75) containing 100 µm Fe-EDTA and
200 µm ferrozine (3-[2-Pyridyl]-5,6-diphenyl-1,2,4-triazine-
4′,4″-disulfonic acid sodium salt) for 3 h at 24°C under dark
conditions, and the resulting chromophore was measured at
562 nm (E = 27.9 mm
−1
cm
−1
) (Stookey, 1970). Root Fe(III)
reduction capacity was also visually demonstrated by fixing
the roots of intact plants in 1% agar (w : v) containing the
same chemical composition used in the ferrozine test.
Measurement of leaf chlorophyll and iron
concentrations
Changes in chlorophyll (SPAD value) were measured on the
youngest expanded leaves before harvest using a chlorophyll meter
(Minolta SPAD-502, Japan). Leaves were dried, ground, and
digested using a microwave digestion system (MarsExpress;
CEM Corp., Matthews, NC, USA) before analysing for Fe
by inductively coupled plasma optical emission spectrometry
(ICP-OES) (Vista-Pro Axial; Varian Pty Ltd, Mulgrave,
Australia).
Measurement of shikimic acid and soluble sugars
Shikimic acid concentrations were measured in apical parts of
roots (3–4 cm) and in young leaves and shoot tips according
to a modification of the method of Cromartie & Polge
(2002). Fresh leaf and root tissues were extracted in 0.25 n
HCl at a ratio of 1 : 10 (w : v) and then centrifuged at
15 000 g for 15 min at + 4°C. The resulting supernatant was
further diluted with 0.25
N HCl at a ratio of 1 : 10 (v : v) and
directly used in the colorimetric assay. Aliquots of 200 µl of
1 : 10 diluted samples were mixed with 400 µl of reaction
solution (a solution of 0.25% periodic acid and 0.25% Na
meta-periodate) and incubated at room temperature for 1 h
to oxidize the shikimic acid in the samples. Finally 400 µl of
quenching solution (0.6 m NaOH and 0.22 m Na
2
SO
3
) was
added to form a yellow chromophore, which correlates directly
with the amount of shikimic acid in the aqueous solution.
Shikimic acid concentration was quantified by measuring the
absorbance of the assay samples at 380 nm by using a standard
curve of 0–100 µm shikimic acid (S5375; Sigma, St Louis,
MO, USA) and presented on a fresh weight basis.
Apical root parts (generally 3–4 cm) were used for analysis
of soluble sugars by using the anthrone method (Yemn & Wills,
1954). Fresh root samples were extracted with 80% ethanol
and centrifuged at 15 000 g for 10 min. The supernatant
obtained was treated with the anthrone solution (7.73 mm
anthrone in 28 n H
2
SO
4
) and incubated in a 90°C water bath
for 20 min. After cooling to room temperature, absorbance of
the green chromophore was measured at 600 nm and the con-
centration of soluble sugars was quantified by using d-glucose
as a standard.
NAD(P)H oxidation
Apical root parts were also used for measurement of NAD(P)H
oxidizing capacity. Fresh root tissues were ground in 25 mm
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)
buffer (pH 7.5) and centrifuged at 15 000 g for 15 min at
+4°C. The supernatant obtained was used for measurement of
NAD(P)H oxidation at 340 nm (E = 6.2 mm
−1
cm
−1
), and
the activity was calculated as nmol of NAD(P)H oxidized per
minute and gram of fresh weight.
For the statistical treatments, see legends of the tables and
figures.
Results
When compared with Fe-sufficient control plants (i.e. 100 µm
Fe), the shoot and root growth of plants with the low Fe
treatment were only slightly reduced after 12-d of growth in
nutrient solution (Table 1). As expected, Fe-sufficient plants
had higher levels of Fe and chlorophyll (SPAD values) than
the Fe-deficient plants. Consistent with reduced SPAD values,
plants receiving the low Fe treatment developed leaf chlorosis
on newly developing leaves. A clear reduction in the pH value
of the nutrient solution of the Fe-deficient plants began at day
10 and by day 12, the pH of the nutrient solution was 4.2
compared with 6.8 for plants supplied with sufficient Fe
(Table 1).
With the onset of leaf chlorosis, glyphosate was sprayed on
shoots at different rates (i.e. 0.32, 0.95 or 1.89 mm). Shoot
and root growth were not affected by increasing glyphosate
applications (Table 2). The selected drift treatments of glyphosate
also had no influence on chlorophyll concentration within a
24 h period (Table 2). However, there was a severe inhibition
of ferric reductase activity of the roots (Fig. 1). Glyphosate
depressed ferric reductase activity of Fe-deficient plants by
nearly 50% within 6 h after the glyphosate treatment (Fig. 1a).
Ferric reductase activity was even more severely inhibited at
12 h and 24 h after the glyphosate treatment, and was lower
than in Fe-sufficient control plants. The glyphosate-dependent
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902
decreases in ferric reductase activity were also dose-dependent
so that as the rate of glyphosate increased from 0 to 1.89 mm
there was a sharp, proportional decrease in reductase activity
of the roots (Fig. 1b). Application of glyphosate as low as
0.32 mm appeared to inhibit reductase activity, but this effect
was not significant (Fig. 1b).
The high sensitivity of ferric reductase to foliar-applied
glyphosate was confirmed using a visual staining technique.
The formation of red colour on and around roots caused by
the formation of Fe(II)-ferrozine complex as result of Fe(III)
reduction was clearly inhibited by increasing rates of glyphosate
(Fig. 2).
Acidification of the growth medium is another reaction of
plants to Fe deficiency. The solution pH of Fe-deficient plants
without glyphosate dropped from an initial pH of 5.5 to pH
4.2, while Fe-sufficient control plants increased the pH to 6.8.
In contrast to ferric reductase, solution pH was not consist-
ently changed in Fe-deficient plants treated with glyphosate
under the experimental conditions used (data not shown).
Shikimate increased fourfold in young leaves and threefold in
apical parts of roots within 6 h after the glyphosate applica-
tion compared with the control. Leaf and root concentrations
of shikimate were even higher at 24 h after the glyphosate
treatment (Fig. 3a). Shikimate accumulation in roots and leaves
had a distinct dose-dependency. As glyphosate increased from
0.32 mm to 1.89 mm, shikimate in young leaves increased
from 0.9 µmol g
−1
FW to 11.5 µmol g
−1
FW and from
0.3 µmol g
−1
FW to 4.6µmolg
−1
FW in roots (Fig. 3b).
Apical parts of roots were also used to determine sugar con-
centrations and NADPH oxidation. Iron-deficient plants had
Table 1 Shoot and root dry matter
production, iron (Fe) concentration,
chlorophyll level in primary leaves and
nutrient solution pH of sunflower plants
(Helianthus annuus cv. TR-3080) grown
with a sufficient (control, 100 µ
M Fe-
ethylenediaminetetraacetic acid (Fe-EDTA))
or a deficient Fe supply (0.2 µ
M Fe-EDTA
for the first 6 d and then without further
Fe treatment).
Dry matter production Fe concentration
Chlorophyll
(SPAD) Solution pH
Shoot Root Shoot Root
(mg per plant) (µg Fe g
−1
DW)
Control 244 ± 33 64 ± 11 221 ± 48 1595 ± 130 34.4 ± 1.2 6.8 ± 0.1
Fe deficiency 196 ± 23 52 ± 10 41 ± 862± 17 16.4 ± 2.9 4.2 ± 0.1
Results are means ± SD of six independent replications.
Fig. 1 Time- (a) and concentration-
dependent (b) inhibition of ferric reductase
activity (means ± SD, n = 6) in iron (Fe)-
deficient sunflower plants (Helianthus annuus
cv. TR-3080) as influenced by glyphosate
(Glyp) treatments. Time-dependent changes
(a) in ferric reductase activity were measured
at 6 h, 12 h and 24 h after 1.89 m
M
glyphosate treatment, whereas
concentration-dependent changes
(b) were measured at 24 h after 0.32, 0.95
and 1.89 m
M glyphosate treatments
(corresponding to 1, 3 and 6% of the
recommended rate for weed control,
respectively). Plants were grown with
sufficient (+Fe, 100 µ
M Fe-
ethylenediaminetetraacetic acid (Fe-EDTA))
or deficient (−Fe) supply of Fe for 12 d in
nutrient solution in a glasshouse. Vertical bar
indicates least significant difference (LSD
0.05
)
at P = 0.05 probability level.
© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 899–906
Research 903
a higher concentration of soluble sugars (Fig. 4a) and tended
to show a greater capacity for NADPH oxidation (Fig. 4b) in
roots when compared with Fe-sufficient plants. Applying
glyphosate at increasing rates tended to increase both sugar
concentrations and NADPH oxidation in the roots (Fig. 4).
Discussion
The present study documents the high sensitivity of ferric
reductase in sunflower roots to foliar-applied glyphosate (Fig. 1).
The inhibitory effect of glyphosate on ferric reductase occurred
at very low, subherbicidal concentrations of glyphosate (i.e. 1–
6% of the widely recommended glyphosate dose for weed
control) corresponding to reported field drift rates of glyphosate
(Ellis et al., 2003; Koger et al., 2005; Bellaloui et al., 2006).
Foliar application of glyphosate at 1.89 mm (e.g. 6% of the
widely recommended glyphosate dose for weed control)
resulted in almost 50% inhibition of reductase activity within
6 h and nearly complete inhibition 24 h after exposure to
glyphosate (Fig. 1). Such substantial inhibition of ferric
reductase occurred before any adverse effects of glyphosate on
shoot or root growth were observed (Table 2).
The mechanism for such substantial and rapid inhibition
of ferric reductase activity by glyphosate was not studied, but
could be related to a sudden shortage of energy needed to
maintain root growth and initiate reductase activity. However,
this was not confirmed by root concentrations of soluble
sugars, which were not influenced by the glyphosate treatments
(Fig. 4a). Both NADPH and NADH have been suggested as
the source of electrons for the reduction of Fe(III) to Fe(II) in
roots of different plant species (Sijmons et al., 1984; Schmidt
& Schuck, 1996; Schmidt, 1999). Glyphosate may affect
the flow of electrons to Fe(III) by interfering with NAD(P)H
oxidation. However, our results, showed that glyphosate did
not interact with NADPH oxidation, and even tended to increase
it (Fig. 4b). Similar findings, as shown in Fig. 4b for NADPH,
were also determined for NADH (data not shown). The results
obtained from root extracts appear to exclude the possibility
that a shortage of sugars or any impairment in NAD(P)H oxida-
tion capacity of root cells was responsible for glyphosate-
induced inhibition of ferric reductase activity. However, further
Fig. 2 Visualization of root iron (Fe) reduction
in sunflower plants (Helianthus annuus cv.
TR-3080) using the ferrozine test in agar
medium. The ferrozine test was performed
24 h after 0.32, 0.95 and 1.89 m
M glyphosate
(Glyp) treatments (corresponding to 1, 3
and 6% of the recommended rate for weed
control, respectively). Plants were grown
with sufficient (+Fe, 100 µ
M Fe-
ethylenediaminetetraacetic acid (Fe-EDTA))
or deficient (−Fe) supply of Fe for 12 d in
nutrient solution in a glasshouse.
Table 2 Shoot and root dry matter production and chlorophyll
concentration of sunflower plants (Helianthus annuus cv. TR-3080)
grown in nutrient solution with a sufficient (+Fe, 100 µ
M Fe-
ethylenediaminetetraacetic acid (Fe-EDTA)) or deficient Fe supply
for 12 d under glasshouse conditions
Treatments
Dry matter production
Chlorophyll
(SPAD)
Shoot Root
(mg per plant)
Control (+Fe −Glyp) 226 ± 30 61 ± 13 39.2 ± 1.9
−Fe −Glyp 208 ± 16 58 ± 8 21.2 ± 1.0
−Fe +Glyp 0.32 m
M 202 ± 16 55 ± 8 21.3 ± 0.7
−Fe +Glyp 0.95 m
M 208 ± 15 56 ± 11 21.7 ± 1.4
−Fe +Glyp 1.89 m
M 194 ± 23 53 ± 9 21.8 ± 2.3
LSD
0.05
24 12 2.3
Glyphosate was sprayed when plants were 11 d old, and harvesting
of plants was realized 24 h after glyphosate application. Results are
means ± SD of six independent replications (LSD
0.05
: least significant
difference at P = 0.05 probability level).
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Research
904
detailed studies are needed to elucidate the mechanism of
inhibition of ferric reductase enzyme by glyphosate.
The rapid reduction in ferric reductase activity following the
glyphosate treatment (Fig. 1), suggests that glyphosate or its
degradation products may form insoluble stable Fe-complexes
that are not useful or not available for reduction by ferric
reductase. Glyphosate has been reported to possess a high affinity
and chelating capacity for Fe and other metals, resulting in the
formation of poorly soluble glyphosate–metal complexes or
insoluble precipitates (Motekaitis & Martell, 1985; Subramaniam
& Hoggard, 1988; Barja et al., 2001; Barrett & McBride,
2005). Formation of such insoluble complexes of glyphosate
with Fe or Mn is a major reason for the loss of herbicidal effec-
tiveness when glyphosate is prepared in ‘hard water’ containing
metals (Bernards et al., 2005). These results suggest that glypho-
sate or its degradation product(s) can diminish the availability
of Fe(III) for the reductase enzyme by forming insoluble com-
plexes. Recent reports of marked decreases in root uptake and
root-to-shoot transport of Fe in sunflower plants were also
attributed to the Fe-complexing ability of glyphosate (Eker
et al., 2006). Alternatively, glyphosate may inhibit reductase
activity directly through an unknown mechanism.
As mentioned earlier, high shikimate concentrations in
glyphosate-treated plant tissues are a good indicator of phyto-
toxicity due to inhibition of EPSPS. Foliar-applied glyphosate
is rapidly translocated to actively growing tomato and spinach
shoots and roots, and accounts for up to 16% of the dry
weight of these tissues (Schulz et al., 1990). In the current
study with sunflower plants, foliar applied glyphosate was also
translocated rapidly and accumulated in meristematic tissues
such as apical root parts. Shikimate concentrations in the apical
parts of roots increased fourfold within 6 h after glyphosate
Fig. 3 Time- (a) and concentration-
dependent (b) changes in shikimate
concentrations (means ± SD, n = 6) in shoots
and roots of iron (Fe)-deficient sunflower
plants (Helianthus annuus cv. TR-3080) as
influenced by glyphosate (Glyp) applications.
Time-dependent changes (a) in shikimate
concentration were measured at 6 h, 12 h
and 24 h after 1.89 m
M glyphosate
treatment, whereas concentration-dependent
changes (b) were measured at 24 h after
0.32, 0.95 and 1.89 m
M glyphosate
treatments (corresponding to 1, 3 and 6%
of the recommended rate for weed control,
respectively). Plants were grown with
sufficient (+Fe, 100 µ
M Fe-
ethylenediaminetetraacetic acid (Fe-EDTA))
or deficient (−Fe) supply of Fe for 12 d in
nutrient solution in a glasshouse. Vertical bar
indicates least significant difference (LSD
0.05
)
at P = 0.05 probability level.
© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 899–906
Research 905
treatment (Fig. 3a). Assuming that root and leaf water content
is 90%, it is estimated that as much as 12 mm shikimic acid
accumulated in young shoots and 5 mm in roots (Fig. 3a).
Apical roots are the plant parts in which ferric reductase is
particularly localized and expressed under Fe deficiency
(Marschner et al., 1986; Marschner & Römheld, 1994). High
expression of ferric reductase activity in root tips can also
be seen in Fig. 2. The colocalization of ferric reductase and
glyphosate in the same root parts facilitates the rapid interac-
tion of glyphosate with ferric reductase. Similar to ferric reductase,
nitrogenase and nitrate reductase activities (both Fe-containing
enzymes) in soybean plants are significantly depressed after
foliar application of glyphosate (Zablotowicz & Reddy, 2004;
Bellaloui et al., 2006). Glyphosate, at 1.25 mm, resulted in a
33% reduction in nitrogenase activity within 24 h (De Maria
et al., 2006); the reason for the decrease is unrelated to protein
damage or a shortage of carbohydrates (energy). Complexing
of the protein bound Fe with glyphosate might explain the
high sensitivity of such Fe-containing enzymes to glyphosate.
Young active nodules are also important accumulation sites of
glyphosate (Bellaloui et al., 2006; De Maria et al., 2006).
Although acidification of the rhizosphere by Fe-deficiency-
induced H
+
release from roots is a characteristic of Strategy-I
plants to stress (Römheld & Marschner, 1986), these results
showed that ferric reductase activity was more sensitive to
glyphosate than was the release of protons from roots into
solution. Apparently, glyphosate does not interfere with the
proton pumping ATPase activity of root cells for 24 h after the
treatment. New experiments should be designed to follow H
+
efflux from intact roots after glyphosate treatment. There are,
however, controversial results in the literature on the effects
of glyphosate on ATPase activity in different cell systems
(Lockau & Pfeffer, 1982; Lopez-Brana et al., 1984; Peixoto,
2005 and references therein).
In conclusion, the results presented in this study showing
that glyphosate is especially inhibitory to ferric reductase
complement the recently published report (Eker et al., 2006)
that glyphosate exerts a strong inhibitory influence on ferric
reductase activity of Fe-deficient roots and impairs the uptake
and translocation of Fe in plants. These impairments could
be a major reason for the increasingly observed Fe deficiency
chlorosis in cropping systems associated with widespread
glyphosate usage as reported for different parts of the USA
(Franzen et al., 2003; Jolley et al., 2004). Such strong inter-
ference of glyphosate with root uptake and root-to-shoot
transport of Fe in crop plants may represent a potential threat
to human and animal nutrition because of possible reduction
of Fe in edible plants parts (e.g. seed/grain).
Acknowledgements
We thank Prof. Don Huber (Purdue University, IN, USA)
and Ernest A. Kirkby (Leeds University, UK) for constructive
comments on the manuscript and correction of the English
text.
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- "The results of this present study was in agreement with Neumann et al. (2006) who demonstrated that glyphosate herbicide applied exclusively to glyphosate-resistance (GR) soybean leaves; impaired Mn uptake of non-GR sunflower seedlings cultivated simultaneously suggested an inhibition of micronutrient uptake by root. Similarly, hydroponic experiments demonstrated that even low levels of glyphosate caused a pronounced decline in acquisition, root uptake and root-to-shoot translocation of radiolabeled Fe and Mn in non-GR sunflower (Ozturk et al. 2008; Eker et al. 2006). In case of macronutrient Ca and Mg significantly lower difference was observed in all doses after all days than control. "
- "Previous studies indicated that some herbicides can interfere with plant nutrition and in particular with zinc (Zn), copper (Cu), manganese (Mn) and iron (Fe) acquisition (Eker et al., 2006; Osborne et al., 1993; Rengel and Wheal, 1997). For instance, it has been demonstrated that frequent applications of glyphosate leads to the development of clear Fe chlorosis symptoms in treated crops (Bellaloui et al., 2009; Ozturk et al., 2008). The alteration of Fe uptake capacity of plants is of great importance, and of evident scientific interest, since Fe together with nitrogen (N) and phosphorous (P), is the most yield limiting crop nutrient in the world (Schachtman et al., 1998; Zhang et al., 2010). "
[Show abstract] [Hide abstract] ABSTRACT: One of the main purposes of modern agriculture is to raise crop production by means of a proper application of herbicides. Nonetheless, the use of herbicides is causing some concerns associated to their persistence and accumulation in the environment. Notwithstanding the relevance of these aspects, little or no attention has been paid to the interferences that these chemicals and their residues can exert on iron (Fe) mineral nutrition of plants. To this purpose, we studied the effect of terbuthylazine (TBA) on the Fe-acquisition processes of Fe-deficient barley plants, by investigating some aspects related to phytosiderophores (PS) exudation and sulfur (S) metabolism. Results showed that plant growth, chlorophyll content expressed as SPAD index, PS release and the expression of genes involved in their secretion, uptake and biosynthesis were negatively affected. This response was associated with reduced cysteine concentrations, and it suggests that the TBA interferences on Fe-acquisition in barley are the consequence of the induced changes in S metabolism.- "Ferric reductase is required for iron absorption in the human gut. Glyphosate reduced the activity of ferric reductase in plants by 50% within six hours of treatment [68], so it might have a similar effect in mammals. Evidence from plants supports the idea that glyphosate may directly interfere with heme synthesis. "
[Show abstract] [Hide abstract] ABSTRACT: Many neurological diseases, including autism, depression, dementia, anxiety disorder and Par-kinson's disease, are associated with abnormal sleep patterns, which are directly linked to pineal gland dysfunction. The pineal gland is highly susceptible to environmental toxicants. Two pervasive substances in modern industrialized nations are aluminum and glyphosate, the active ingredient in the herbicide, Roundup ®. In this paper, we show how these two toxicants work synergis-tically to induce neurological damage. Glyphosate disrupts gut bacteria, leading to an overgrowth of Clostridium difficile. Its toxic product, p-cresol, is linked to autism in both human and mouse models. p-Cresol enhances uptake of aluminum via transferrin. Anemia, a result of both aluminum disruption of heme and impaired heme synthesis by glyphosate, leads to hypoxia, which induces increased pineal gland transferrin synthesis. Premature birth is associated with hypoxic stress and with substantial increased risk to the subsequent development of autism, linking hypoxia to autism. Glyphosate chelates aluminum, allowing ingested aluminum to bypass the gut barrier. This leads to anemia-induced hypoxia, promoting neurotoxicity and damaging the pineal gland. Both glyphosate and aluminum disrupt cytochrome P450 enzymes, which are involved in mela-tonin metabolism. Furthermore, melatonin is derived from tryptophan, whose synthesis in plants and microbes is blocked by glyphosate. We also demonstrate a plausible role for vitamin D3 dys-biosis in impaired gut function and impaired serotonin synthesis. This paper proposes that impaired sulfate supply to the brain mediates the damage induced by the synergistic action of aluminum and glyphosate on the pineal gland and related midbrain nuclei.
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