hormesis with herbcides: glyphosate as a case study stephen o. duke, joanna bajsa, scott r. baerson...
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Hormesis with Herbcides:Glyphosate as a Case Study
Stephen O. Duke, Joanna Bajsa, Scott R. BaersonNatural Products Utilization Research Unit, Agricultural Research
Service, United States Department of Agriculture, University, MS, USA
Nina CedergreenUniv. Copenhagen, Tåstrup, Denmark
Edivaldo VeliniUniv. São Paulo State, Botucatu, Brazil
Solvent control was 1% DMSO (by vol)
Naptalam5-27-2004
log concentration (M)
0.001 0.01 0.1 1 10 100 1000 10000
Lem
na g
row
th (
%)
0
100
200
300
400
500
600
700
Day 5 Day 7
Effects of naptalam on L. paucicostata growth
Inhibits auxin translocation
OH
O
O
N
Scopoletin D11-9-01
Concentration (M)
0.1 1 10 100 1000 10000
Lem
na g
row
th (
% in
7 d
ays)
0
100
200
300
400
500
600
O
O
OHO
Effects of scopoletin on L. paucicostata growth
Unknown mode of action
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
P
HO
N
OH
O OHH+
_
Highly translocatable
Co-translocates with sucrose to metabolic sinks (i.e., meristems).
Gougler and Geiger, Plant Physiol. 68: 668 (1981)
Glyphosate (kg ae/ha)
0.001 0.01 0.1 1 10 100Sho
ot d
ry w
eigh
t (%
of
nont
reat
ed c
ontr
ol)
0
20
40
60
80
100
120
Non-GR HBKC 5025GR Asgrow 4603
~ 50 X resistance factor
Nandula et al., J. Agric. Food Chem. – in press (2007)
Year
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
% o
f cul
tiva
ted
lan
d
0
20
40
60
80
100
SoybeanCotton
"Morgan, Sandy" <[email protected]>
>80% of transgenic crops were glyphosate resistant in 2006
Other uses
Sugarcane ripener
Copyright ©2006 by the National Academy of Sciences
Funke, Todd et al. (2006) Proc. Natl. Acad. Sci. USA 103, 13010-13015
Inhibition of EPSP synthase by glyphosate
Copyright ©2006 by the National Academy of Sciences
Funke, Todd et al. (2006) Proc. Natl. Acad. Sci. USA 103, 13010-13015
Three-dimensional structure of CP4 EPSP synthase
Mechanism of action of glyphosatePhosphoenolpyruvate + Erythrose 4-phosphate
DAHP synthase
Shikimate 3-phosphateEPSP synthase
Chorismate
Anthranilate
Tryptophan
Arogenate
Phenylalanine and Tyrosine
Glyphosate
Plant defenses PhytoalexinsLignin
IAASecondary products
Shikimate pathway
Protein
Phosphoenolpyruvate + Erythrose 4-phosphate
DAHP synthase
Shikimate 3-phosphateEPSP synthase
Chorismate
Anthranilate
Tryptophan
Arogenate
Phenylalanine and Tyrosine
Glyphosate
Plant defenses PhytoalexinsLignin
IAASecondary products
Shikimate pathway
Protein
Shikimate
Protocatechuate andother benzoates
More carbon from other pathways flows into the shikimate pathway
Salicylate?
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, 228-232.
Schabenberger, O. et al., (1999) Statistical test for hormesis and effective dosage in herbicide dose-response. Agron. J.
100
135.8
196.7
112.1106.3
55.5
87.6
0
50
100
150
200
0 1.8 3.6 7.2 18 36 72
Glyphosate (g/ha)
% o
f con
trol
Increase in leaf dry weight 55 days after application of glyphosate to
Pinus sp.
10086.6
244
140.3
94.5 93.6
0
50
100
150
200
250
0 3.6 7.2 18 36 72
Glyphosate (g/ha)
% o
f con
trol
Increase in leaf area 30 days after application of glyphosate to coffee
(Caffea arabica)
100
121.9127.5
167
153.8147.2
70.5
0
50
100
150
0 1.8 3.6 7.2 18 36 72
Glyphosate (g/ha)
% o
f con
trol
Increase in leaf area 25 days after application of glyphosate to
Sorghum bicolor
0.4 1.3 3.7 6.7 12.7 14.1 18.1
80.4
97.4
134
236.1
0
40
80
120
160
200
240
0 1.8 3.6 7.2 18 36 72 180 360 720 1440
Glyphosate (g/ha)
ug s
hiki
mat
e/g
fr w
tShikimate in leaves, 20 days after application of glyphosate to
Sorghum bicolor
hormesis
1
19.521.4 21.9 23.3
25.4
35.9
51.6
61
65.967.8
0
20
40
60
0 1.8 3.6 7.2 18 36 72 180 360 720 1440
Glyphosate (g/ha)
ug s
hiki
mat
e/g
fr w
tShikimate in leaves, 30 days after application of glyphosate to coffee
plants
hormesis
Glyphosate Control
Glyphosate Control 1.8 g/ha
3.6 g/ha
7.2 g/ha
Glyphosate Control
Eucalyptus grandis
0
20
40
60
80
100
120
140
160
180
200
220
0 1 10 100 1000
glyphosate rates (g / ha)
% o
f th
e ch
eck
Root Weights
Leaf Weights
Stem Weights
Total Weights
Leaf Area
0.6 3.3 5.79.7
14.7 14.1 15.9
60.467.4
74
167.1
0
50
100
150
0 1.8 3.6 7.2 18 36 72 180 360 720 1440
Glyphosate (g/ha)
ug s
hiki
mat
e/g
fr w
tShikimate in leaves, 45 days after application of glyphosate to
Eucalyptus grandis
hormesis
Conventional soybean
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
1 10 100 1000
glyphosate rates (g/ha)
Dry
we
igh
ts (
% o
f th
e c
he
ck)
Root
Shoot
Total
Growth of glyphosate-resistant transgenic soybean
The application of rates from 1.8 to 720.0 g/ha did not affect the growth of glyphosate-resistant transgenic soybean
g / ha Root Shoot Total
0 100.00 100.00 100.00
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
Coefficient of variation (%) 19.05 16.75 12.91
F values:
1.15ns 0.82ns 1.25ns
Glyphosate rates Dry weights ( % of the check)
Treatments or rates
NS: Not significant; (*): p<0.05; (**): p<0.01
Effects of glyphosate on growth of L. paucicostata
Potential mechanism(s?) of hormesis by glyphosate
Phosphoenolpyruvate + Erythrose 4-phosphate
DAHP synthase
Shikimate 3-phosphateEPSP synthase
Chorismate
Anthranilate
Tryptophan
Arogenate
Phenylalanine and Tyrosine
Flavonoids +Isoflavanoids
LigninsHydroxycinnamates
Alkaloids
Indole alkaloids
Acridone alkaloids
Indole phytoalexins
Indole glucosinolates
1
2
Barley measured 4 days after spraying
glyphosate (g/ha)
10-day old barley treated and then evaluated 4 days later
Leaf length (cm/plant) Shoot length (cm/plant)
Glyphosate (g/ha) Glyphosate (g/ha)
10-day old barley treated and then evaluated 4 days later
Root dry wt increase (g/plant) Shoot dry wt increase (g/plant)
Glyphosate (g/ha) Glyphosate (g/ha)
10-day old barley treated and then evaluated 4 days later
Plant dry weight increase (g/plant) Relative growth rate (d-1)
Glyphosate (g/ha) Glyphosate (g/ha)
Used Affymetrix barley chip
Not a whole genome chip
25,500 genes
Microarray experiment
12-day-old plants sprayed with 5, 10 and 20 g/ha glyphosate
2 8 20 h
RNA samples Growth determined at 5 days after treatment
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 5 10 15 20 25
Series1
g glyphosate/ha
Fre
sh w
eigh
t (g
/pla
nt)
RTPCR of phenyalalaine ammonia-lyase
0
0.5
1
1.5
2
2.5
3
3.5
4
2h 2hcontrol
4h 4hcontrol
8 h 8hcontrol
20h 20hcontrol
PAL transcript
Light off
Light on
RTPCR of DAHPS
0
1
2
3
4
5
6
2h 2hcontrol
4h 4hcontrol
8 h 8hcontrol
20h 20hcontrol
DAHPS transcript
Light offLight on
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 hours1. 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 hours1. 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
stressendomembranecell wallotherunknown
Functional breakdown of affected genes
Further work needed
Cell wall aspects Lignification Anatomical measurements
Photosynthetic aspects (Cedergreen) Hormonal aspects