Introduction to Glyphosate Mode of Action and Bioassay Calculations
Introduction to Glyphosate Mode of Action and Bioassay Calculations
Introduction to Glyphosate Mode of Action and Bioassay Calculations
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<strong>Introduction</strong> <strong>to</strong> <strong>Glyphosate</strong><br />
<strong>Mode</strong> <strong>of</strong> <strong>Action</strong> <strong>and</strong><br />
<strong>Bioassay</strong> <strong>Calculations</strong><br />
Michael Lovelace, Ph.D.<br />
Agronomist, USDA, AMS<br />
Seed Regula<strong>to</strong>ry <strong>and</strong><br />
Testing Branch<br />
Gas<strong>to</strong>nia, NC<br />
http://www.ams.usda.gov/lsg/seed.htm<br />
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The Seed Regula<strong>to</strong>ry<br />
<strong>and</strong> Testing Branch<br />
AMS Lab <strong>and</strong> Office Complex, Gas<strong>to</strong>nia, NC<br />
• Enforces interstate commerce provisions <strong>of</strong><br />
the Federal Seed Act (FSA).<br />
• Provides the global seed community with<br />
services that promote the truthful marketing <strong>of</strong><br />
seed. Agricultural Marketing Service<br />
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The Seed Regula<strong>to</strong>ry<br />
<strong>and</strong> Testing Branch<br />
AMS Lab <strong>and</strong> Office Complex, Gas<strong>to</strong>nia, NC<br />
• ISTA accredited<br />
• ASL accreditation pending<br />
• ISO 9001 compliant<br />
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SRTB Tests Performed<br />
• Purity<br />
• Germination<br />
• Noxious weed seed<br />
• Moisture / Conductivity<br />
• Species identification<br />
• Plant pathology<br />
• Variety verification<br />
– Varietal purity / Presence <strong>of</strong><br />
biotech derived seed<br />
• <strong>Bioassay</strong><br />
• Protein based<br />
• DNA based<br />
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New Test Development
New Test Development<br />
• Where do we begin?<br />
• Important things <strong>to</strong> know:<br />
– Previous literature / studies<br />
– Background on the crop<br />
– Background on the herbicide<br />
– Knowledge <strong>of</strong> experimental design /<br />
statistics<br />
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Biochemistry Review<br />
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• What are:<br />
– Amino Acids?<br />
– Proteins?<br />
– Enzymes?
Amino Acids<br />
• An amino acid is any molecule<br />
containing amino <strong>and</strong> carboxylic<br />
acid functional groups.<br />
• AAs are the basic structural<br />
building units <strong>of</strong> proteins.<br />
• AA form short polymer chains<br />
called peptides or polypeptides,<br />
which in turn form proteins.<br />
• The coding sequences <strong>of</strong> genes<br />
determines the AA sequences <strong>of</strong><br />
almost all naturally occuring<br />
proteins though transcription<br />
<strong>and</strong> translation.<br />
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Proteins<br />
• Protein comes from the Greek word<br />
"pro<strong>to</strong>s“, meaning "<strong>of</strong> primary<br />
importance". Proteins are essential <strong>to</strong><br />
the structure <strong>and</strong> function <strong>of</strong> all living<br />
cells.<br />
• Many proteins are enzymes or subunits<br />
<strong>of</strong> enzymes. Others are important for<br />
structural or mechanical roles,<br />
signalling functions, immune response,<br />
s<strong>to</strong>rage, <strong>and</strong> transport.<br />
• In nutrition, proteins are broken down<br />
through digestion <strong>to</strong> provide amino<br />
acids for an organism, including those<br />
the organism may not be able <strong>to</strong><br />
synthesise itself.<br />
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Enzymes<br />
• An enzyme is a protein that catalyzes, or<br />
speeds up, a chemical reaction.<br />
• Enzymes are essential <strong>to</strong> life because<br />
most chemical reactions in biological<br />
cells occur <strong>to</strong>o slowly, or lead <strong>to</strong><br />
different products, without enzymes.<br />
• Enzymes lower the reaction activation<br />
energy, thus speeding up the reaction.<br />
They remain unaltered by the reaction<br />
<strong>and</strong>, therefore, can continue function.<br />
• Enzyme activity can be affected by other<br />
molecules. Inhibi<strong>to</strong>rs decrease or<br />
abolish enzyme activity, while activa<strong>to</strong>rs<br />
increase activity. Aspirin inhibits the<br />
COX enzymes that produce the<br />
inflamation messenger prostagl<strong>and</strong>in,<br />
thus suppressing pain <strong>and</strong> inflammation.<br />
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H<br />
Amino Acid Synthesis<br />
NH 3 +<br />
R<br />
COO -<br />
• Carbon skele<strong>to</strong>ns come from<br />
intermediates <strong>of</strong> glycolysis, the<br />
pen<strong>to</strong>se phosphate pathway, or<br />
citric acid cycle. These<br />
skele<strong>to</strong>ns contain carboxylic<br />
acid groups.<br />
• Nitrogen is assimilated in<strong>to</strong><br />
amino acids by way <strong>of</strong> glutamate<br />
<strong>and</strong> glutamine.<br />
• Synthesize using five different<br />
precursors via various<br />
pathways.<br />
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Alanine (Ala) Arginine (Arg)<br />
Aspargine (Asn) Aspartate (Asp)<br />
Cysteine (Cys)<br />
Glutamate (Glu) Glutamine (Gln)<br />
Methionine (Met)<br />
Branched Chain<br />
Amino Acids<br />
Aromatic<br />
Amino Acids<br />
Isoleucine (Ile)<br />
Amino Acids<br />
Proline (Pro)<br />
Glycine (Gly)<br />
Leucine (Leu)<br />
Histidine (His)<br />
Serine (Ser) Threonine (Thr)<br />
Valine (Val)<br />
Phenylalanine (Phe) Tyrosine (Tyr) Tryp<strong>to</strong>phan (Trp)<br />
Lysine (Lys)<br />
Inhibited by<br />
ALS Inhibiting<br />
Herbicides<br />
Inhibited by<br />
<strong>Glyphosate</strong>
D-Erythrose-4-Phosphate<br />
H 2 O<br />
P i<br />
7-Phospho-2-dehydro-<br />
3-deoxy-Darabinoheptulosonate<br />
NAD +<br />
NADH,<br />
H + , P i<br />
Shikimate Pathway<br />
Production <strong>of</strong> Aromatic Amino Acids<br />
3-Dehydroquinate<br />
Phosphoenolpyruvate<br />
O<br />
O -<br />
Enzyme<br />
complex<br />
O<br />
O- O- P<br />
O<br />
Co++<br />
3-Dehydroquinate<br />
synthase<br />
3-Dehydroquinate<br />
dehydratase<br />
H 2 O<br />
3-Dehydroshikimate<br />
Chorismate<br />
Shikimate<br />
dehydrogenase<br />
NADPH,<br />
H +<br />
NADP +<br />
3-Phospho-5-enoylpyruvylshikimate<br />
Chorismate<br />
synthase<br />
P i<br />
Shikimate<br />
3-Phospho-5enoylpyruvylshikimate<br />
synthase<br />
Mg++<br />
Shikimate<br />
kinase<br />
3-Phosphoshikimate<br />
(S3P)<br />
ATP<br />
ADP<br />
P i<br />
PEP
Chorismate<br />
Precursor for Aromatic Amino Acids<br />
Chorismate<br />
Prephenate Anthranilate<br />
Phenylalanine Tyrosine Tryp<strong>to</strong>phan
Influence <strong>of</strong> <strong>Glyphosate</strong> in Plants<br />
<strong>Glyphosate</strong> molecules<br />
in a spray droplet
Chloroplast<br />
O<br />
O -<br />
O<br />
O- O- P<br />
O<br />
Chorismate<br />
3-Phospho-5enoylpyruvylshikimate<br />
synthase<br />
Shikimate<br />
x
O -<br />
O -<br />
O<br />
P<br />
O<br />
O -<br />
P<br />
O -<br />
O<br />
PEP<br />
N<br />
Competitive Inhibition<br />
COO -<br />
<strong>Glyphosate</strong><br />
COO -<br />
EPSPS<br />
open conformation closed conformation
How does resistance work?<br />
Site <strong>of</strong> herbicide action<br />
in a conventional plant<br />
O -<br />
O<br />
P<br />
O -<br />
N<br />
<strong>Glyphosate</strong><br />
Interaction results in a conformational change that will not allow PEP <strong>to</strong> bind.<br />
Site <strong>of</strong> herbicide action<br />
in a transgenic plant<br />
O -<br />
O<br />
P<br />
O -<br />
N<br />
COO -<br />
<strong>Glyphosate</strong><br />
Changing a base pair that codes for an amino acid in the site <strong>of</strong> herbicide action<br />
changes the localized charges <strong>of</strong> the enzyme, thus inhibiting glyphosate binding.<br />
This permits PEP <strong>to</strong> bind <strong>and</strong> the shikimate pathway <strong>to</strong> function normally.<br />
COO -
How does this knowledge relate<br />
<strong>to</strong> bioassays?<br />
• <strong>Glyphosate</strong> activity occurs in<br />
the chloroplast. A light cycle<br />
must be present for<br />
glyphosate <strong>to</strong> have an affect.<br />
• Seeds do not have<br />
chloroplasts, so a certain<br />
level <strong>of</strong> plant development is<br />
necessary in order for the<br />
bioassay <strong>to</strong> work.<br />
• This is the reason we have<br />
some growth <strong>of</strong> the plants<br />
before growth is inhibited.<br />
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Summary<br />
• <strong>Glyphosate</strong> translocates in<strong>to</strong><br />
the chlorophyll.<br />
• In the chlorophyll, glyphosate<br />
inhibits the EPSPS enzyme,<br />
thus inhibiting production <strong>of</strong><br />
aromatic amino acids.<br />
• Plants die due <strong>to</strong> starvation.<br />
• In transgenic plants, a mutated<br />
EPSPS enzyme is insensitive <strong>to</strong><br />
glyphosate, thus allowing PEP<br />
<strong>to</strong> bind normally.<br />
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Time <strong>to</strong> Change Gears!!<br />
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<strong>Bioassay</strong> Setup<br />
• Acquire sample seeds from purity<br />
test <strong>and</strong> count out the number <strong>of</strong><br />
seeds needed <strong>to</strong> conduct the test.<br />
– Sample in question<br />
• Roundup Treatment<br />
• Water Check<br />
– Know susceptible sample<br />
• Roundup Treatment<br />
• Water Check<br />
– Known resistant sample<br />
• Roundup Treatment<br />
• Water Check<br />
• Place the seeds in a petri-dishes <strong>and</strong><br />
correctly label the seeds for each<br />
treatment.
Prepare Working Solution<br />
• A working solution is the herbicide<br />
mixture used for seed soaking or<br />
wetting the <strong>to</strong>wels.<br />
• Determine the concentration <strong>of</strong> the<br />
working solution from the test<br />
directions <strong>and</strong> gather information from<br />
pesticide label.<br />
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Preparation <strong>of</strong> Herbicide<br />
Working Solutions<br />
Prepare calculations for<br />
your herbicide working<br />
solution.<br />
Calculate concentration in<br />
PPM (Parts Per Million).<br />
or<br />
Calculate concentration in<br />
molarity (g / L).<br />
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Short Quiz<br />
• What is a PPM?<br />
• How do you calculate PPM?<br />
• What are the units <strong>of</strong> PPM?<br />
• Why is PPM unit-less?
Gathering Pertinent Information<br />
• Locate the area on the<br />
container that contains the<br />
pertinent information<br />
• Identify the active ingredients<br />
<strong>and</strong> corresponding<br />
concentrations.<br />
• Know what information is<br />
useful.<br />
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How much glyphosate is in 1<br />
Liter <strong>of</strong> Roundup?<br />
• Container indicates 50.2%<br />
glyphosate.<br />
• 50% <strong>of</strong> 1 liter is 500 ml, thus<br />
there are 500 ml <strong>of</strong><br />
glyphosate in 1 L.<br />
• The container indicates there<br />
are 600 ml <strong>of</strong> glyphosate in 1<br />
L.<br />
• Where did we go wrong?<br />
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Why Not Calculate<br />
Based on Percentages?<br />
• Existing bioassay methods<br />
indicate concentration<br />
calculations should be<br />
based on percentage.<br />
• This can lead <strong>to</strong> problems<br />
because the label usually<br />
does not specify if<br />
percentages are based on<br />
weight or volume.<br />
• Why does it matter?<br />
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Importance <strong>of</strong> Percentage Type<br />
50% V / V (volumetric)<br />
Solvent<br />
1.4 g / cm 3<br />
glyphosate<br />
1.7 g / cm 3<br />
If the<br />
solution is<br />
mixed on a<br />
volume<br />
basis,<br />
there will<br />
be about<br />
10% more<br />
glyphosate<br />
on a weight<br />
basis.<br />
50% g / g (gravimetric)<br />
Solvent<br />
1.4 g / cm 3<br />
glyphosate<br />
1.7 g / cm 3<br />
If the<br />
solution is<br />
mixed on a<br />
weight<br />
basis,<br />
there will<br />
be about<br />
10% more<br />
solvent on<br />
a volume<br />
basis.<br />
If we assume that the percentage <strong>of</strong> glyphosate is 50% by volume<br />
<strong>and</strong> glyphosate is actually 50% by weight, we would be adding 10%<br />
less roundup <strong>to</strong> our working solution than we would anticipate.
Why Not Calculate<br />
Based on Percentages?<br />
• So, is it volumetric or<br />
gravimetric??<br />
• We don’t always know!!<br />
• Instead, use g ai / Liter<br />
or lb ai / gal<br />
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Calculating Working Solutions<br />
Determine the concentration <strong>of</strong><br />
your working solution <strong>and</strong> how<br />
much you will need for the<br />
experiment.<br />
Examine herbicide container <strong>to</strong><br />
establish concentration <strong>of</strong> s<strong>to</strong>ck<br />
solution.<br />
Calculate the dilution <strong>of</strong> your<br />
s<strong>to</strong>ck solution <strong>to</strong> make your<br />
desired working solution.<br />
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1000 PPM;<br />
1 Liter<br />
600 g / Liter<br />
???
Calculating Working Solutions in PPM<br />
1,000 parts<br />
999,000 parts<br />
1 part<br />
999 parts<br />
Roundup Ultra Max = 600 g ai<br />
1000 ml<br />
600 g ai<br />
1000 ml<br />
= 1 g ai<br />
cross multiply<br />
X<br />
Water 1 ml = 1 g<br />
1 g<br />
999 g<br />
= =<br />
=<br />
X =<br />
1 ml<br />
999 ml<br />
Must dilute roundup <strong>to</strong> acquire<br />
desired concentration.<br />
1 g ai X 1000 ml<br />
600 g ai<br />
X = 1.67 ml<br />
Add the 1.67 ml <strong>of</strong> herbicide solution <strong>to</strong> 998.33 ml <strong>of</strong> water <strong>to</strong> make a<br />
1000 PPM working solution.<br />
Are we done?? Not so fast!! There is still more <strong>to</strong> consider!!<br />
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Calculating Working Solutions in PPM<br />
• Lets revisit the label.<br />
• The label indicates glyphosate is in<br />
the form <strong>of</strong> isopropylamine salt.<br />
• Why is this important?<br />
• The isopropylamine salt portion <strong>of</strong><br />
the formulation has no herbicidal<br />
activity. Furthermore, different<br />
formulations <strong>of</strong> glyphosate have<br />
different salts.<br />
• In order <strong>to</strong> avoid any confusion,<br />
our calculations need reflect the<br />
amount <strong>of</strong> glyphosate free acid<br />
(portion without the salt) in the<br />
solution.
Formulation Comparisons<br />
HO<br />
O<br />
O<br />
N P<br />
O -<br />
<strong>Glyphosate</strong> Free Acid (169 g / mole)<br />
+ H3 N<br />
Isopropylamine Salt (59 g / mole)<br />
• <strong>Glyphosate</strong> free acid = 169 g / mole<br />
• IPA salt <strong>of</strong> glyphosate = 228 g / mole<br />
• DAM salt <strong>of</strong> glyphosate = 201 g / mole<br />
• TMS salt <strong>of</strong> glyphosate = 244 g / mole
Calculating Working Solutions in PPM<br />
1,000 parts<br />
999,000 parts<br />
1 part<br />
999 parts<br />
1 g<br />
999 g<br />
= =<br />
=<br />
Roundup = 600 g IPA salt <strong>of</strong> glyphosate<br />
Ultra Max 1000 ml herbicide solution<br />
Roundup = 0.445 g glyphosate free acid<br />
Ultra Max ml herbicide solution<br />
0.445 g ai<br />
1 ml herb<br />
= 1 g ai<br />
X<br />
cross multiply<br />
Water 1 ml = 1 g<br />
X =<br />
1 g ai X 1 ml<br />
0.445 g ai<br />
1 ml<br />
999 ml<br />
X = 2.25 ml herb<br />
Due <strong>to</strong> the different densities <strong>of</strong> water <strong>and</strong> herbicide solution, it may not<br />
be appropriate <strong>to</strong> add 2.25 ml <strong>of</strong> herbicide solution <strong>to</strong> 997.75 ml <strong>of</strong> water.<br />
Instead, it would be more appropriate <strong>to</strong> tare a beaker on a balance.<br />
Add about half the quantity <strong>of</strong> water desired. Add your herbicide<br />
solution. Add water <strong>to</strong> the beaker until the scale reads 1000 grams.<br />
X<br />
169 g glyphosate free acid<br />
228 g IPA salt <strong>of</strong> glyphosate<br />
Herb solution 1 ml ≠ 1 g
Use <strong>of</strong> Molarity<br />
• Use g ai / Liter or lb ai / gal<br />
• Use molecular weight (169.07)<br />
<strong>of</strong> the glyphosate molecule.<br />
• Percentage, density <strong>of</strong><br />
solution, <strong>and</strong> makeup <strong>of</strong> inert<br />
ingredients is irrelevant in<br />
calculation.<br />
• <strong>Calculations</strong> are accepted SI<br />
units, whereas PPM is not.<br />
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Conclusions<br />
• Things are not always as<br />
easy as they seem.<br />
• We need <strong>to</strong> rethink the way<br />
we do our calculations.<br />
• A common worksheet may<br />
st<strong>and</strong>ardize our herbicide<br />
solutions.<br />
• Molarity is a great<br />
alternative because there is<br />
no confusion with units.
Questions or Comments