Hormesis with Herbcides:
Glyphosate as a Case Study
Stephen O. Duke, Joanna Bajsa, Scott R. Baerson
Natural Products Utilization Research Unit, Agricultural Research
Service, United States Department of Agriculture, University, MS, USA
Nina Cedergreen
Univ. Copenhagen, Tåstrup, Denmark
Edivaldo Velini
Univ. São Paulo State, Botucatu, Brazil
700
Inhibits auxin translocation
O
600
N
OH
Lemna growth (%)
500
O
400
300
200
100
0
Effects of naptalam on L. paucicostata growth
0.001
0.01
0.1
1
10
log concentration (M)
Day 5
Day 7
Solvent control was 1% DMSO (by vol)
100
1000
10000
600
Unknown mode of action
Lemna growth (% in 7 days)
500
400
300
200
O
100
HO
O
O
0
Effects of scopoletin on L. paucicostata growth
0.1
1
10
100
Concentration (M)
1000
10000
N-phosphonomethylglycine
(glyphosate)
 Simple zwitterionic
amino acid
 First synthesized in
1950 by Henri Martin
(Cilag Co.) for unknown
purposes
 First tested for
herbicidal activity by
Monsanto Agric.
Products in 1970.
O
_ O
P
HO
+
H
H
O
N
OH
Highly translocatable
 Co-translocates with
sucrose to metabolic
sinks (i.e., meristems).
Gougler and Geiger, Plant
Physiol. 68: 668 (1981)
Shoot dry weight (% of nontreated control)
120
~ 50 X resistance factor
100
80
60
40
20
0
0.001
Non-GR HBKC 5025
GR Asgrow 4603
0.01
0.1
1
10
100
Glyphosate (kg ae/ha)
Nandula et al., J. Agric. Food Chem. – in press (2007)
100
% of cultivated land
80
Soybean
Cotton
60
40
20
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Year
"Morgan, Sandy" <Sandy.Morgan@ARS.USDA.GOV>
>80% of transgenic crops were
glyphosate resistant in 2006
Other uses
 Sugarcane ripener
Inhibition of EPSP synthase by glyphosate
Funke, Todd et al. (2006) Proc. Natl. Acad. Sci. USA 103, 13010-13015
Copyright ©2006 by the National Academy of Sciences
Three-dimensional structure of CP4 EPSP synthase
Funke, Todd et al. (2006) Proc. Natl. Acad. Sci. USA 103, 13010-13015
Copyright ©2006 by the National Academy of Sciences
Mechanism of action of glyphosate
Phosphoenolpyruvate + Erythrose 4-phosphate
DAHP synthase
Shikimate
pathway
Shikimate 3-phosphate
EPSP synthase
Glyphosate
Chorismate
Anthranilate
Arogenate
IAA
Secondary
products
Phenylalanine and Tyrosine
Tryptophan
Protein
Plant defenses
Phytoalexins
Lignin
More carbon from other pathways flows into the shikimate pathway
Phosphoenolpyruvate + Erythrose 4-phosphate
Salicylate?
DAHP synthase
Protocatechuate and
other benzoates
Shikimate
pathway
Shikimate 3-phosphate
EPSP synthase
Glyphosate
Shikimate
Chorismate
Anthranilate
Arogenate
IAA
Secondary
products
Phenylalanine and Tyrosine
Tryptophan
Protein
Plant defenses
Phytoalexins
Lignin
Previous report of hormesis with
glphosate
 Wagner, R. et al., (2003) Phytotoxic activity
of root absorbed glyphosate in corn seedlings
(Zea mays L.). Weed Biol. Manag. 3, 228232.
 Schabenberger, O. et al., (1999) Statistical
test for hormesis and effective dosage in
herbicide dose-response. Agron. J.
Increase in leaf dry weight 55 days after application of glyphosate to
Pinus sp.
196.7
% of control
200
150
135.8
112.1
100
100
106.3
87.6
55.5
50
0
0
1.8
3.6
7.2
18
Glyphosate (g/ha)
36
72
Increase in leaf area 30 days after application of glyphosate to coffee
(Caffea arabica)
244
250
% of control
200
140.3
150
100
100
86.6
94.5
93.6
36
72
50
0
0
3.6
7.2
18
Glyphosate (g/ha)
Increase in leaf area 25 days after application of glyphosate to
Sorghum bicolor
167
153.8
% of control
150
121.9
100
147.2
127.5
100
70.5
50
0
0
1.8
3.6
7.2
18
Glyphosate (g/ha)
36
72
Shikimate in leaves, 20 days after application of glyphosate to
Sorghum bicolor
236.1
ug shikimate/g fr wt
240
hormesis
200
160
134
120
97.4
80.4
80
40
0
0.4
1.3
3.7
6.7
0
1.8
3.6
7.2
12.7
14.1
18.1
18
36
72
180
Glyphosate (g/ha)
360
720 1440
Shikimate in leaves, 30 days after application of glyphosate to coffee
plants
65.9
67.8
ug shikimate/g fr wt
61
60
51.6
hormesis
40
35.9
19.5
20
21.4
21.9
3.6
7.2
23.3
25.4
1
0
0
1.8
18
36
72
180
Glyphosate (g/ha)
360
720 1440
Glyphosate
Control
1.8 g/ha
Glyphosate
7.2 g/ha
3.6 g/ha
Glyphosate
Control
Control
Eucalyptus
grandis
220
Root Weights
% of the check
200
Leaf Weights
180
Stem Weights
160
Total Weights
Leaf Area
140
120
100
80
60
40
20
0
0
1
10
glyphosate rates (g / ha)
100
1000
Shikimate in leaves, 45 days after application of glyphosate to
Eucalyptus grandis
ug shikimate/g fr wt
167.1
150
100
60.4
hormesis
67.4
74
50
0
0.6
3.3
5.7
0
1.8
3.6
9.7
7.2
14.7
14.1
15.9
18
36
72
180
Glyphosate (g/ha)
360
720 1440
Conventional soybean
Dry weights (% of the check)
140
130
120
Root
Shoot
110
100
90
80
70
Total
60
50
40
30
20
10
0
-10
1
10
100
glyphosate rates (g/ha)
1000
Growth of glyphosate-resistant transgenic soybean
Glyphosate rates
g / ha
Dry weights ( % of the check)
0
Root
100.00
Shoot
100.00
Total
100.00
1.8
88.81
110.12
105.14
3.6
113.58
106.33
108.02
7.2
90.45
102.23
99.47
18
105.53
118.71
115.63
36
103.46
102.91
103.04
72
87.88
94.76
93.15
180
95.21
96.47
96.18
360
99.87
107.98
106.09
720
86.68
100.67
97.40
19.05
16.75
12.91
1.15ns
0.82ns
1.25ns
Coefficient of variation (%)
F values:
Treatments or rates
NS: Not significant; (*): p<0.05; (**): p<0.01
The application of rates from 1.8 to 720.0 g/ha did not affect the growth of
glyphosate-resistant transgenic soybean
Effects of glyphosate on growth of L. paucicostata
Potential mechanism(s?) of hormesis by glyphosate
Phosphoenolpyruvate + Erythrose 4-phosphate
DAHP synthase
Shikimate 3-phosphate
EPSP synthase
1
Acridone alkaloids
Chorismate
Indole
alkaloids
Indole phytoalexins
Anthranilate
Arogenate
Phenylalanine and Tyrosine
Tryptophan Hydroxycinnamates
Lignins
Indole glucosinolates
2
Alkaloids
Flavonoids +
Isoflavanoids
Barley measured 4 days after spraying
glyphosate (g/ha)
10-day old barley treated and then
evaluated 4 days later
Leaf length (cm/plant)
Glyphosate (g/ha)
Shoot length (cm/plant)
Glyphosate (g/ha)
10-day old barley treated and then
evaluated 4 days later
Root dry wt increase (g/plant)
Glyphosate (g/ha)
Shoot dry wt increase (g/plant)
Glyphosate (g/ha)
10-day old barley treated and then
evaluated 4 days later
Plant dry weight increase (g/plant)
Glyphosate (g/ha)
Relative growth rate (d-1)
Glyphosate (g/ha)
Used Affymetrix barley chip
 Not a whole genome
chip
 25,500 genes
Microarray experiment
RNA samples
2
8
20 h
12-day-old plants sprayed with 5, 10 and 20 g/ha glyphosate
Growth determined
at 5 days after
treatment
1.6
Fresh weight (g/plant)
1.4
1.2
1
0.8
Series1
0.6
0.4
0.2
0
0
5
10
g glyphosate/ha
15
20
25
RTPCR of phenyalalaine ammonia-lyase
4
Light off
3.5
3
PAL transcript
2.5
Light on
2
1.5
1
0.5
0
2h
2h
control
4h
4h
control
8h
8h
control
20h
20h
control
RTPCR of DAHPS
6
5
4
DAHPS transcript
3
Light off
Light on
2
1
0
2h
2h
control
4h
4h
control
8h
8h
control
20h
20h
control
Very little in terms of a dramatic
response
 2h upregulated - 12 genes
 8h upregulated - 2 genes
 20h upregulated - 3 genes
 2h downregulated - 0 genes,
 8h downregulated - 0 genes,
 20h downregulated - 3 genes.
2 hours
1. Cytochrome P450-like protein - 3.4 X
2. Putative cell wall protein – 3.1 X
3. Very-long-chain fatty acid condensing enzyme – 3.3 X
4. putative GDSL-motif lipase/hydrolase-like protein – 3.1 X
5. Structural protein – 4.1 X
6. Putative proline-rich protein – 3.1 X
7. Unknown – 4.7 X
8. Histone H2A.9 - wheat dbj|BAA07279.1| protein H2A – 6.1 X
9. 9e-37 expressed protein; protein – 3.9 X
10. Unknown – 4.7 X
11. Cysteine proteinase – 8.4 X
12. Thionine Osthi1 – 14.7 X
8 hours
1. Cysteine proteinase – 8 X
2. En/Spm-like transposon protein – 7X
20 hours
Upregulated
1. Unknown (different than at 2 h) – 4 X
2. Ethylene-responsive transcriptional coactivator-like protein – 2.3 X
3. En/Spm-like transposon protein – 2 X
Downregulated
1.
Polygalacturonase-inhibiting protein - -1.5X
2.
Unknown (different than up-regulated none genes) - -1.1
3.
GA-regulated protein, GASA precursor - - 1.25
Functional breakdown of affected genes
stress
endomembrane
cell wall
other
unknown
Further work needed
 Cell wall aspects


Lignification
Anatomical measurements
 Photosynthetic aspects (Cedergreen)
 Hormonal aspects