Hormesis with Herbcides: Glyphosate as a Case Study Stephen O. Duke, Joanna Bajsa, Scott R. Baerson Natural Products Utilization Research Unit, Agricultural.

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