New Insight in the Mode of Action of Glufosinate

Franck Dayan, Professor, Agricultural Biology, Colorado State University Author

12/02/2021 Added

8 Plays

Description

The effect of glufosinate on glutamine synthetase and ammonia accumulation have been known for many years. However, our understanding of the contact action of this herbicide could not be accounted for by these effects. Our detailed investigation elucidated the biochemical mechanism leading to the rapid ROS-driven burn-down effect of glufosinate. During this research, we stumbled upon a physiological connection between glufosinate and PPO inhibitors that led to new synergistic herbicide mixtures.

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[00:00:00.760]The following presentation
[00:00:02.220]is part of the Agronomy and Horticulture Seminar Series
[00:00:05.810]at the University of Nebraska, Lincoln.
[00:00:08.750]Good afternoon.
[00:00:09.950]Good afternoon, everybody.
[00:00:11.930]We do have a small group of people here in the room
[00:00:15.470]and we have probably what?
[00:00:16.610]About 20 plus over the Zoom.
[00:00:20.690]So, for those of you who don't know me,
[00:00:23.690]I'm Steve Knezevic I'm a UNL lead scientist
[00:00:25.750]who's been around for 20 some years.
[00:00:28.440]And that it's my pleasure
[00:00:29.780]to actually introduce Dr. Franck Dayan.
[00:00:34.400]Franck comes from Colorado State University.
[00:00:39.450]He and I are about the same generation.
[00:00:42.590]He's got a very interesting background,
[00:00:44.760]both from the academic standpoint and the personal.
[00:00:48.190]As you guys can see,
[00:00:50.260]this is the way they'll spell Franck in France.
[00:00:53.850]So Franck was born and raised in Southern France,
[00:00:59.440]actually in the general area of Marseille.
[00:01:02.740]So those of you who like geography,
[00:01:04.410]you can pull it up on Google
[00:01:05.537]and you will see Marseille, the beautiful city
[00:01:08.610]right on the coast of Mediterranean,
[00:01:12.410]which is one of my favorites seas in this world anyway.
[00:01:16.130]And then he came to United States a long time ago,
[00:01:21.210]is like 35 years plus,
[00:01:24.190]and then he did most of his schooling here.
[00:01:26.910]And then he had almost a 20 year position
[00:01:33.510]with you USDA and Mississippi.
[00:01:35.960]And then he had joined
[00:01:39.733]or Colorado State.
[00:01:41.303]I almost said K state, I'm a K state graduate.
[00:01:44.370]So that's a little blurb on, I apologize on my side.
[00:01:48.300]He joined Colorado State about five years ago.
[00:01:51.600]With that, Franck again, thank you for coming to Nebraska
[00:01:56.110]and the floor is yours.
[00:01:58.170]Thank you. Yeah.
[00:01:59.380]Thank you, Stevan.
[00:02:01.110]It's a pleasure to be here
[00:02:02.720]and welcome to all of you online.
[00:02:05.110]I know with the COVID, we're getting more and more used
[00:02:07.890]to having attendance online.
[00:02:11.300]It just feels a little bit different
[00:02:13.900]from our perspective here.
[00:02:15.940]So I have quite a bit to cover
[00:02:18.270]so I wanted to go ahead and start.
[00:02:20.580]My presentation will be in three sections
[00:02:23.000]and I might take a pause between each section
[00:02:26.830]to have questions,
[00:02:27.780]but otherwise we can have the questions at the end as well.
[00:02:31.220]And what I wanna talk to you about today
[00:02:33.950]is about the mode of action of glufosinate
[00:02:37.050]because glufosinate has been around for a long time
[00:02:39.570]and there was lots of literature
[00:02:42.220]on its target site, glutamine sythetase,
[00:02:45.930]but really I was not convinced that the way people explained
[00:02:49.750]how glufosinate killed the plants
[00:02:52.350]was actually how it did it.
[00:02:53.530]So I had a grad student on a project
[00:02:56.990]that was funded by Bayer initially, and then BSF
[00:03:00.220]after BSF purchased glufosinate from Bayer.
[00:03:03.890]So we'll talk about the discovery
[00:03:05.490]and use of glufosinate very briefly,
[00:03:09.210]and a bit more about how it works
[00:03:10.900]and then some outcome from our research,
[00:03:13.330]which is we discovered a really interesting synergy
[00:03:16.010]between PPO herbicides and glufosinate.
[00:03:18.720]So first the discovery,
[00:03:20.080]and I'm gonna go pretty quickly on this
[00:03:21.520]'cause I don't wanna spend too much time.
[00:03:24.553]Glufosinate turns out to be the actually
[00:03:26.220]the only true natural product herbicide,
[00:03:29.450]because it's actually derived from Bialaphos.
[00:03:33.390]Bialaphos is something that was produced by
[00:03:36.110]some streptomyces species
[00:03:39.450]that can be used as a natural herbicide.
[00:03:42.091]And when it's put on the plant is metabolize
[00:03:44.210]and what it releases is L-phosphinothricin
[00:03:48.440]and that is a structure of glufosinate.
[00:03:50.890]The main difference is that,
[00:03:53.540]okay a little bit about where it falls in,
[00:03:57.410]like glufosinate is group 10
[00:04:00.260]and it's an amino acid biosynthesis inhibitor inhibits
[00:04:03.560]the biosynthesis of glutamine from glutamate.
[00:04:07.900]But because of what we discovered,
[00:04:10.310]this is not how it kills the plant,
[00:04:12.700]it's actually a light dependent herbicide,
[00:04:14.500]so the new extract map positions glufosinate right here
[00:04:19.000]under the light activation of ROS.
[00:04:21.260]So it's pretty much more
[00:04:22.430]like a photosynthesis related herbicide
[00:04:26.050]than an amino acid biosynthesis herbicides,
[00:04:28.490]and I'll tell you more about this in a minute.
[00:04:31.860]Something about it is a contact herbicide on the left
[00:04:34.820]is a control on the right is glufosinate treated
[00:04:38.930]and what you will see that within a few hours,
[00:04:42.520]those leaves are gonna burn down.
[00:04:44.720]So it acts very quickly and unlike most other,
[00:04:48.700]amino acid biosynthesis inhibitors, like the ALS
[00:04:51.347]and EPSPS that tend to be slower acting
[00:04:54.540]because you have to deplete the amino acids.
[00:04:58.410]Obviously within 24 hours, our plant will be dying here,
[00:05:03.380]and this is how we...
[00:05:04.600]I always thought glufosinate is doing something else
[00:05:07.390]than just inhibiting amino acid biosynthesis, okay?
[00:05:11.080]The main thing about glufosinate is like it's a mixture
[00:05:13.800]of the D and the L form of phosphinothricin,
[00:05:18.230]that's a natural product that is not a natural product.
[00:05:21.020]So it cannot be commercialized as a organic herbicide
[00:05:24.010]because it contains some of the D-phosphinothricin,
[00:05:27.650]but structurally it's actually derived
[00:05:29.210]from a natural product.
[00:05:32.220]We know we have transgenic genes for resistance,
[00:05:34.790]the bar and the pat genes.
[00:05:36.620]And you can see in this field, this one survived
[00:05:38.760]and these of course, wound roundup ready
[00:05:41.060]and Xtend both die
[00:05:42.880]because they don't have the pat or the bar gene in there.
[00:05:46.780]And what that gene does is it takes glufosinate
[00:05:50.870]and it's an acetylation,
[00:05:53.200]so it adds an acetyl group to the amine group
[00:05:55.127]and you end up with N-Acetyl-phosphinothricin
[00:05:58.055]and this is no longer herbicidal.
[00:06:01.800]We know that it's non-selective broad-spectrum,
[00:06:06.480]we need to have good coverage,
[00:06:09.170]the plants needs to be less than four inches tall,
[00:06:12.900]a lot of water, at least 15 gallons per acre
[00:06:17.140]and we need sunlight for it to work really well,
[00:06:21.050]and coverage is very important.
[00:06:22.820]And that's the deal with glufosinate
[00:06:24.830]is that when it works, it works really well,
[00:06:27.090]but sometimes it just fails.
[00:06:29.160]And part of our work was try to understand
[00:06:31.920]why it would fail, see if we could come up with methods
[00:06:34.840]to make it work better.
[00:06:37.170]So here's an example
[00:06:40.210]of experiments we've done
[00:06:45.110]with glufosinate,
[00:06:46.227]and on the left, that's the time of day effect.
[00:06:50.550]When we sprayed during the daylight
[00:06:51.970]you get very good control,
[00:06:53.110]when he sprayed at dusk, right before night,
[00:06:57.700]we lose a lot of the efficacy.
[00:06:59.180]You can see the violin plot,
[00:07:00.990]there's a lot of drug from 100 down to 25 right here.
[00:07:04.630]So we lose a lot of the efficacy
[00:07:06.260]by just spraying it later in the day,
[00:07:09.240]and that's also something interesting that we figured out.
[00:07:11.600]Okay, so a foliar absorption is a big factor.
[00:07:15.400]The more glufosinate like most herbicides goes in
[00:07:19.400]the more efficacy we have,
[00:07:21.520]and we looked at different concentrations in nanomolar
[00:07:23.930]and specially linear.
[00:07:25.070]So the more you put, the more goes in the plant.
[00:07:28.880]What's interesting is that there's a big pH effect here
[00:07:33.590]below five, pH five, we have really good absorption,
[00:07:37.360]as soon as you go above 4.5, the absorption goes down a lot.
[00:07:41.800]So the pH or the spray solution is very important as well.
[00:07:46.870]And we know that it's a contact herbicide.
[00:07:49.250]And if you read the literature,
[00:07:51.020]it says that glufosinate does not translocate.
[00:07:54.720]Now what we've done in this experiment
[00:07:56.650]is we either treated this leaf,
[00:07:58.540]I mean, in this part of the leaf or this part of the leaf,
[00:08:01.290]and then we measured how much glufosinate moved.
[00:08:04.630]And so if we treated glufosinate here
[00:08:09.310]about almost 40% of it moved up,
[00:08:14.660]so it does move through the apoplast.
[00:08:17.940]In fact, I've done greenhouse experiments
[00:08:20.010]that if you put in the roots, your whole foliars dies
[00:08:23.070]so that it can be taken up by the roots
[00:08:25.100]and move to the foliage very rapidly.
[00:08:27.730]However, if you do the other way,
[00:08:29.090]if you put glufosinate towards the tip of the leaf,
[00:08:33.610]then we have very little translocation.
[00:08:35.680]So there's no simplastic translocation at all.
[00:08:39.540]And it's interesting because if you look at the structure
[00:08:41.660]of glufosinate, it's like a natural product,
[00:08:44.850]is very much like glyphosate
[00:08:46.590]but there's really no reason why it would not move,
[00:08:49.150]but it just doesn't move through the simplast.
[00:08:54.735]So with those response,
[00:08:56.520]because we know some species are more sensitive than others,
[00:08:59.790]you can see these five different curves for ryegrass
[00:09:03.710]horseweed, kochia, Johnsongrass, and Palmer.
[00:09:07.040]So this is visual injury by dose,
[00:09:11.510]and this is visual injury over time.
[00:09:14.270]And the one that will, of course, were the least sensitive
[00:09:18.860]had the least evolution of injury is slower over time,
[00:09:24.000]but plants like Palmer are very, very sensitive.
[00:09:26.800]We get almost 100% injury within 48 hours of application.
[00:09:35.463]So as I said, if you look at this
[00:09:38.060]there's differences in sensitivity to glufosinate,
[00:09:42.950]and if you look at the amount of glufosinate uptake,
[00:09:45.930]the two more tolerant one had a lot less absorption
[00:09:49.890]than the three more sensitive one.
[00:09:52.120]So again, that's a good correlation
[00:09:53.890]that efficacy is proportional
[00:09:56.660]to the absorption of the herbicide,
[00:10:00.000]so it's proportional to the absorption.
[00:10:02.940]So I just wanna kind of pause right here
[00:10:05.320]because I went pretty fast,
[00:10:06.630]but I wanna make sure that I can spend more time
[00:10:09.060]on the mode of action.
[00:10:10.000]So essentially real recap,
[00:10:13.130]so glufosinate is actually derived from a natural product.
[00:10:16.880]And if you could commercialize the L-glufosinate alone
[00:10:20.900]that could be an organic herbicide,
[00:10:22.550]that would be non-selective, very effective herbicide.
[00:10:27.620]And we've talked to AAMRI
[00:10:30.182]and why they would not accept
[00:10:34.870]the natural product as a herbicide,
[00:10:37.090]when in fact they accept all kinds of crazy chemistry
[00:10:40.670]in organic agriculture,
[00:10:42.550]and they would not give us a straight answer.
[00:10:44.430]And what we believe is that if you spray the natural product
[00:10:48.040]or the mixture of the D and the L,
[00:10:51.130]analytically, it would be very difficult to tell
[00:10:53.080]whether somebody sprayed the synthetic form
[00:10:55.820]or the natural product form,
[00:10:57.560]and that's maybe why they're not accepting it.
[00:11:02.280]Non-selective is very good, but sometimes it fails,
[00:11:05.460]so now I'll talk more about this,
[00:11:07.150]maybe why sometimes it fails,
[00:11:09.350]and a key factor is the absorption
[00:11:12.160]the efficacy of the herbicide is proportional
[00:11:13.850]to the absorption of the herbicide in the plant.
[00:11:16.880]So a little bit more about how it works,
[00:11:19.220]because this is interesting.
[00:11:20.570]When we think about ammonia, the nitrogen cycle in plant,
[00:11:27.240]nitrate is absorbed through the soil
[00:11:30.170]with nitrate transporters.
[00:11:33.120]There's a nitrate reductase that converts it to nitrite,
[00:11:37.420]nitrite moves into the chloroplasts,
[00:11:39.200]there's a nitrite reductase which makes ammonia
[00:11:42.210]and ammonia is used by glutamine synthetase
[00:11:46.050]and combine it with glutamate to make glutamine.
[00:11:49.152]So that's basically how ammonia enters the plant
[00:11:51.750]into amino acids.
[00:11:53.250]And then there's this beautiful enzymes B-gal
[00:11:57.360]that can take glutamine
[00:11:59.150]and what's called ketoglutarate right here
[00:12:02.050]and out of these two molecules it makes two glutamate.
[00:12:05.120]So essentially it's a cycle
[00:12:06.980]that can generate a lot of glutamate
[00:12:09.130]and then glutamate is a center amino acid
[00:12:12.990]that serves as a nitrogen donor for most other amino acids.
[00:12:17.000]So then you can then multiply one glutamate
[00:12:19.460]to two glutamate,
[00:12:20.520]and as the cycle goes around and around,
[00:12:23.280]you always end up with an extra glutamate
[00:12:25.750]and then that can come out of the chloroplast
[00:12:28.377]and in the cytosol can be converted to glutamine
[00:12:30.930]and then that can be exported.
[00:12:32.580]So that in the seed of the plant,
[00:12:35.750]there'll be more glutamine than glutamate,
[00:12:37.550]that's the formless translocate in the plant is glutamine.
[00:12:42.620]And this enzyme glutamine sythetase,
[00:12:44.550]so there's two of them,
[00:12:45.500]there's one in the chloroplast and one in the cytosol,
[00:12:48.730]that's the second most abundant protein in the plant leaf.
[00:12:51.820]So it's very important.
[00:12:53.230]The most abundant one being RuBisCO,
[00:12:55.440]the one involved in photosynthesis.
[00:12:57.430]So it's a very important target.
[00:12:59.270]Okay, a couple of structures here,
[00:13:01.440]that's the glutamine sythetase,
[00:13:03.577]that's actually a crystal structure with glufosinate
[00:13:07.800]within its catalytic domain right here.
[00:13:11.240]And the reaction is actually a two-step reaction,
[00:13:14.210]so I wanna spend a little time on this
[00:13:15.740]because the first thing is glutamate reacts with ATP
[00:13:21.550]and you end up with phophoglutamate,
[00:13:23.760]so it's glutamate with a phospho group right here.
[00:13:27.340]And then that is an high-energy molecule
[00:13:30.210]that can then be used for the emanation
[00:13:32.740]and will end up with the glutamine
[00:13:34.790]with a nitrogen over here.
[00:13:36.540]Now the reason why I wanna show you this
[00:13:38.700]is that if you look at these two structures,
[00:13:40.920]that's the intermediate, the gamma glutamyl phosphate
[00:13:44.890]that's phosphorylated glufosinate.
[00:13:47.700]And if you look at the structures,
[00:13:50.020]they are almost identical.
[00:13:52.590]That is why glufosinate works so well,
[00:13:56.120]it competes with this reaction intermediates,
[00:13:59.580]and it's a irreversible inhibitor,
[00:14:01.930]so it completes bonds irreversibly
[00:14:04.897]and that's called a suicide inhibitor,
[00:14:08.020]because it kills the enzyme.
[00:14:09.810]The enzyme activity will never recover,
[00:14:12.570]and that is one of the facts of why it's so efficient.
[00:14:16.120]So we know glufosinate inhibits the conversion
[00:14:19.810]of glutamate to glutamine,
[00:14:21.695]and there was a lot of papers talking about
[00:14:25.270]the inhibition of the enzyme.
[00:14:28.040]And there was a lot of papers about
[00:14:29.750]the fact that that causes ammonia accumulation
[00:14:33.040]and logically people would say,
[00:14:35.450]well, of course, it's an amino acid biosynthesis
[00:14:37.630]that's how it kills the plant,
[00:14:39.520]and ammonia is what's the toxic component
[00:14:43.080]that makes those contact symptoms right here.
[00:14:46.650]And so actually we wanted to test this
[00:14:49.370]and to do this, we used C3 plants
[00:14:52.320]that tend to do more photorespiration
[00:14:55.687]and C4 plants that tend to do less photorespiration.
[00:14:58.897]And the reason why we do that is because glutamine sythetase
[00:15:03.440]is a key enzyme in the photorespiratory pathway.
[00:15:07.910]So this is really interesting
[00:15:09.770]I showed you this figure similar...
[00:15:13.970]Before I will show you phototoxicity or injury,
[00:15:17.490]and as we increase the dose, there's more and more injury
[00:15:20.270]and that's basically the same order
[00:15:22.260]with Palmer being the more sensitive
[00:15:24.610]and ryegrass being in the more tolerant.
[00:15:26.900]So if we measure the enzyme in the plant,
[00:15:31.020]it looks just like injury, it's the same pattern.
[00:15:33.950]What's interesting is if you take these enzymes,
[00:15:36.400]extract them from the plant and do the same experiment,
[00:15:39.830]you get something like this.
[00:15:41.590]When in fact they are all as sensitive to glufosinate
[00:15:45.400]as each other.
[00:15:46.750]So the main difference is that here there's less absorption,
[00:15:49.810]I showed you that earlier, right?
[00:15:51.260]That these plants absorb less,
[00:15:53.670]so there's less inhibition in the plant,
[00:15:55.520]this one absolves more,
[00:15:56.750]there's more inhibition in the plant,
[00:15:58.490]but if you take the enzyme out,
[00:15:59.740]they actually there's no difference
[00:16:00.880]in the enzymes themselves,
[00:16:02.270]they are all sensitive to glufosinate, okay?
[00:16:07.940]Now this is carbon assimilation.
[00:16:11.550]So that's of course, photosynthesis,
[00:16:15.400]we have the light reaction that uses electrons,
[00:16:18.200]and then we have the dark reaction
[00:16:19.800]or the Calvin cycle that converts CO2 into sugars in plants.
[00:16:24.980]And what we found was interesting is that
[00:16:27.900]the C3 plants were more inhibited
[00:16:35.780]than the C4 plants, right here, Palmer, right?
[00:16:40.850](clears throat)
[00:16:42.020]And that's because these plants do more photorespiration
[00:16:46.070]than the C4 plants,
[00:16:48.190]so these plants tend to be more sensitive, but not always.
[00:16:51.850]And so this is a time course,
[00:16:54.200]which is then as we inhibit this,
[00:16:56.650]do we have ammonia accumulation?
[00:16:58.380]And the more sensitive plant have more ammonia accumulation
[00:17:01.443]than the resistant or the tolerant plant.
[00:17:04.140]This is all background information
[00:17:05.540]'cause hopefully in the end, it will make sense
[00:17:08.580]that inhibition of carbon assimilation
[00:17:11.410]is not consistent with the phototoxicity
[00:17:13.930]and ammonia accumulation is not consistent either.
[00:17:17.320]I'll show you some more data on that.
[00:17:19.470]So we thought, well, is this symptomology,
[00:17:23.460]the phenotype burned down effect, is it due to ROS?
[00:17:27.110]And this is where the studies got really interesting
[00:17:29.860]because of course we could monitor that with dyes.
[00:17:33.350]As you treat glufosinate on plants,
[00:17:35.930]you get a lot of ROS over time.
[00:17:37.780]This is hydrogen superoxide on top
[00:17:40.300]and that's hydrogen peroxide on the bottom
[00:17:42.710]and so that would be the pattern over time
[00:17:44.730]within less than 12 hours,
[00:17:47.110]we basically max out all the ROS
[00:17:49.400]and then these ROSes we know can cause lipid peroxidation.
[00:17:54.450]And so lipid peroxidation is when reactive oxygen in species
[00:17:59.180]react with lipids of membranes, it breaks down the membrane,
[00:18:02.810]and one way to measure that
[00:18:04.670]is by measuring the breakdown product,
[00:18:07.750]that's called the MDA, malondialdehyde.
[00:18:10.829]So not only then we had increase in ROS,
[00:18:15.150]but we could monitor degradation of membranes,
[00:18:18.660]and it was also very quick,
[00:18:19.810]within eight hours you get a lot of damage.
[00:18:22.520]So clearly then the contact activity of glufosinate,
[00:18:27.360]even though it's related
[00:18:28.470]to inhibition of glutamine sythetase
[00:18:30.950]and it's related to ammonia accumulation,
[00:18:33.170]what's killing the plant is the ROS
[00:18:35.110]and the rapid lipid peroxidation of the membrane.
[00:18:39.880]So that people would say
[00:18:41.730]that this was what killing the plant,
[00:18:43.640]where we actually concluded from our studies
[00:18:46.320]that these are just a side-effect.
[00:18:48.750]You inhibit glutamine sythetase,
[00:18:50.710]that causes ammonia accumulation
[00:18:52.370]and inhibits a carbon assimilation.
[00:18:55.110]But that's not what's killing the plant,
[00:18:56.890]what's killing the plant is ROS and lipid peroxidation
[00:19:01.170]and that's proportional
[00:19:02.350]to the concentration in the leaf, okay?
[00:19:05.600]So we published all that recently.
[00:19:08.240]And then the question was, well, where do the ROS come from?
[00:19:12.400]Why would inhibiting amino acid biosynthesis cause ROS?
[00:19:18.500]ALS inhibitors don't cause great Ross,
[00:19:25.042]inhibition of EPSPS does not cause great ROS.
[00:19:28.560]We wanted to know what was the connection,
[00:19:32.060]so I'm gonna show you boxes in blue will be increase
[00:19:35.460]and in red will be decrease.
[00:19:36.680]We did what's called a targeted metabolomics.
[00:19:39.570]We look at organic acids in plants
[00:19:41.560]after spraying with glufosinate.
[00:19:44.300]So when we inhibited glufosinate,
[00:19:46.610]to remind you, we are inhibiting that enzyme right here.
[00:19:50.980]What happened is all of these accumulated
[00:19:55.130]and all of these decreased, all right?
[00:19:58.230]So even though the inhibition target is over here,
[00:20:02.350]the consequences are elsewhere,
[00:20:05.080]and clearly the bottleneck is right here, right?
[00:20:08.940]Everything before this step accumulates,
[00:20:11.970]everything after these steps decreases.
[00:20:14.920]So that helped us a lot to understand what was going on,
[00:20:17.440]that even though everybody was looking over here,
[00:20:20.020]really what's going on is over here.
[00:20:23.510]So this accumulates, these decreases, and that's because...
[00:20:28.370]That's actually interesting too
[00:20:29.850]because when you inhibit the step, you will think,
[00:20:32.450]okay, you inhibit glutamine sythetase,
[00:20:34.976]so you should not have any glutamine,
[00:20:36.750]but you should have a lot of glutamate accumulation.
[00:20:39.430]But for some reason that does not happen
[00:20:41.000]because everything that requires glutamate decreases.
[00:20:46.000]So this is where everything happens.
[00:20:48.410]And so that shows the connection of glufosinate,
[00:20:53.170]I mean glutamine synthetase with photorespiration
[00:20:56.700]and all of these metabolites are toxic to RuBisCO.
[00:21:01.970]So I'm gonna step back one more time.
[00:21:04.670]Here's what our current understanding is.
[00:21:06.940]So we have photosynthesis
[00:21:08.760]with PS2, PS1, ATP synthase,
[00:21:13.990]all of that with photosynthesis
[00:21:15.500]is to generate NADPH and ATP.
[00:21:18.460]And I know this is kind of not necessarily
[00:21:21.950]the most popular topic in agronomy
[00:21:24.430]because people tend to do more field work.
[00:21:26.310]I get really excited about pathways
[00:21:28.100]and about chemical processes.
[00:21:30.550]So the whole purpose of photosynthesis
[00:21:33.200]is to use these electrons to make NADPH
[00:21:36.480]and then the protons to make ATP,
[00:21:38.670]and that's used for RuBisCO later on, right?
[00:21:42.180]And what we did is we wanted to test now,
[00:21:45.840]can we connect photosynthesis
[00:21:47.800]to the efficacy of glufosinate?
[00:21:49.930]So we use very small amounts of atrazine
[00:21:52.330]enough to slow down the flow of electron
[00:21:55.220]without killing the plants.
[00:21:56.910]And we use a compound called Dinoseb, that's an uncoupler
[00:22:00.530]which allows electron transport,
[00:22:04.541]but prevents electron transport,
[00:22:06.700]but still have oxygen evolution, and here's what happened.
[00:22:10.910]So as far as visual injury, control no injury,
[00:22:14.460]we use a rate of glufosinate that would cause a low injury,
[00:22:17.340]we didn't wanna kill the plants, right?
[00:22:19.330]So we were aiming for 50%, we achieved about 30% inhibition.
[00:22:23.720]What happens is if you put a small amount of atrazine,
[00:22:26.670]glufosinate loses activity, right?
[00:22:29.503]So that shows that the electrons in photosynthesis
[00:22:33.340]are the source of the ROSes, right?
[00:22:35.460]The ROSes are formed from these electrons,
[00:22:38.280]because if you slow down the flow of electrons,
[00:22:41.030]then you remove the injury.
[00:22:43.310]However, what happens is when you leave it with atrazine,
[00:22:47.300]not only you stop the flow of electrons,
[00:22:49.710]but you stop oxygen evolution.
[00:22:52.010]So there's no oxygen and ROS, what are they?
[00:22:54.740]They are reactive oxygen species.
[00:22:57.650]So if you block both electron and oxygen evolution,
[00:23:02.150]glufosinate loses activity.
[00:23:04.510]Dinoseb blocks electrons, but keeps the oxygen
[00:23:10.170]and you have an increase in efficacy of glufosinate.
[00:23:14.540]So now we found a connection between photosynthesis,
[00:23:17.670]photorespiration and glutamine synthetase, right?
[00:23:21.900]So what happens with ROS?
[00:23:24.470]No ROS in the control,
[00:23:25.870]we're aiming for small amount of injuries,
[00:23:28.670]so you have some ROS with glufosinate.
[00:23:31.070]With atrazine we reduced the amount of ROS
[00:23:33.277]and with Dinoseb we can increase the amount of ROS, okay?
[00:23:37.170]So that gives us clues,
[00:23:38.330]so you can start peeling the onion a little bit.
[00:23:40.460]Okay, this does this, this is not important,
[00:23:42.510]this is very important.
[00:23:44.910]So we then measured electron transport,
[00:23:46.930]and what we show here
[00:23:48.620]is that's the different transport and control.
[00:23:51.110]We use glufosinate, there's a reduction
[00:23:52.980]in electron transport,
[00:23:54.370]and both glyphosinate and atrazine
[00:23:56.550]and this is like very low doses,
[00:23:58.370]I think it's one-twentieth of a dose of atrazine or Dinoseb,
[00:24:03.240]you completely block the flow of electron.
[00:24:05.650]But the difference is,
[00:24:07.980]you block oxygen evolution with atrazine,
[00:24:12.870]no injury, no ROS,
[00:24:15.720]with glufosinate and Dinoseb we recover that injury
[00:24:22.048]and that's because you still have oxygen evolution.
[00:24:24.890]So little by little, we were able to pull things together
[00:24:28.290]that if we could make glufosinate always cause ROS
[00:24:33.660]then you could always have a more consistent way of working.
[00:24:38.540]So the last figures from this section
[00:24:40.670]which was published in photosynthesis research
[00:24:43.660]is the full picture.
[00:24:45.310]That's glutamate to glutamine,
[00:24:49.360]that's a target of a glufosinate, that enzyme.
[00:24:52.810]When we inhibit this,
[00:24:54.410]we show that all of these precursors accumulate
[00:24:57.650]that inhibits RuBisCO.
[00:24:59.243]Phosphoglycolate is an inhibitor of RuBisCO.
[00:25:02.600]So when that happens, then...
[00:25:05.387]Let me back up I went too fast.
[00:25:07.190]Under normal circumstances,
[00:25:09.807]electrons move, you get NADPH and ATP,
[00:25:13.240]you get the Calvin Cycle, you have some RuBisCO,
[00:25:17.480]I mean some photorespiration,
[00:25:19.520]and then the enzyme works just fine right here.
[00:25:21.920]The small amount of excess electrons can form ascorbate,
[00:25:25.800]and I mean, can form reactive oxygen species,
[00:25:28.760]but there's an enzyme that quench those things.
[00:25:31.050]So under normal circumstances, there is no injury.
[00:25:33.800]When we inhibit it, we block all of these pathway.
[00:25:39.600]Do I have that somewhere?
[00:25:40.590]Yeah, so these electrons back up,
[00:25:43.670]we get more reactive oxygen species,
[00:25:46.440]and then ultimately the plant tries to quench the effects
[00:25:50.840]of both the peroxidation and the ascorbate, peroxidase.
[00:25:54.790]They both increase, try to quench this excess ROS,
[00:25:58.360]but when you get too much of it,
[00:26:00.690]it overwhelms the system lipid peroxidation
[00:26:03.337]and you kill the plant.
[00:26:04.850]So this is really how glufosinate works.
[00:26:07.240]It's more complicated than just inhibition
[00:26:09.100]of glutamine synthetase, is all tied to photosynthesis,
[00:26:12.720]and this is why it's moved into the column
[00:26:14.620]of ROS generating herbicides
[00:26:17.150]rather than amino acid biosynthesis inhibitors.
[00:26:20.439]All right, and time-wise, I have until what time?
[00:26:24.943]30 minutes.
[00:26:26.050]30 more minutes.
[00:26:26.883]Okay, so I'm gonna take a pause here
[00:26:28.410]because this was pretty deep, that's pretty intense.
[00:26:31.626]So if anybody online has a question
[00:26:34.500]about the mode of action of glufosinate
[00:26:37.062]because it's pretty complicated.
[00:26:39.193]And if any of you have questions,
[00:26:40.625]I'd like to maybe take a pause
[00:26:41.458]because it's a lot of information.
[00:26:43.680]Yes.
[00:26:45.508]This on a special that you mentioned earlier
[00:26:47.970]kind of the different between the light
[00:26:50.279]kind of when you sprayed those plants during the day
[00:26:53.470]versus during the dawn, especially during the dawn,
[00:26:56.450]there is still sunlight out there.
[00:26:59.270]Can we just say that this is only this one
[00:27:01.419]contributed to these kinds of electron oxidation
[00:27:04.290]that we are getting PS2 system or no?
[00:27:06.503]Yes, yes, so that's a good question.
[00:27:08.620]So the question is the time of day effect of glufosinate
[00:27:13.320]that it's much more active when it's sprayed
[00:27:15.850]in the highlight intensity, hottest part of the day,
[00:27:18.640]rather than at dusk and maybe at dawn,
[00:27:20.850]and is that related to photosynthesis?
[00:27:24.080]Absolutely.
[00:27:25.300]The one thing that has really been puzzling to me is
[00:27:29.530]that if you spray at dusk, so there's not much light,
[00:27:35.010]but what we show,
[00:27:36.370]and I'm not showing you this 'cause I have too much data,
[00:27:38.730]is that we inhibit glutamine sythetase in the dark,
[00:27:42.240]but because there's no light, there's no ROS.
[00:27:44.440]What was not clear to me is why the next day
[00:27:48.290]that you don't get the ROS, right?
[00:27:50.490]So we're actually working on a paper,
[00:27:51.940]we look at the rate of the protein turnover,
[00:27:55.420]so how quickly is plant making new glutamine sythetase?
[00:28:00.040]Because as I mentioned,
[00:28:01.240]the inhibitor is a suicide inhibitor.
[00:28:03.640]It kills the enzyme, it does not dissociate.
[00:28:06.580]So once it's bound, the enzyme is dead.
[00:28:09.020]So you would think if that's the case, the next morning,
[00:28:12.320]all the injuries should happen,
[00:28:13.990]but what happen is during the night,
[00:28:16.030]the synthesis of glutamine sythetase is diurnal,
[00:28:19.090]is actually these cycles with the light.
[00:28:22.730]So that by the next day, the enzymes that are dead are dead,
[00:28:25.600]but the new enzyme is already there
[00:28:27.470]and then you lose the effect.
[00:28:28.620]So the only time you have the contact activity
[00:28:32.800]is when you inhibit the enzyme
[00:28:35.860]while the sun is present.
[00:28:37.990]But if you miss that window, then by the next day,
[00:28:42.310]even though the herbicide is still in the plant
[00:28:44.120]is bound to the enzyme,
[00:28:45.910]the next day there's new enzyme that's not bound
[00:28:47.980]and there's no effect at all.
[00:28:49.560]Oh, much less effect.
[00:28:51.230]So that answers your question?
[00:28:53.360]Okay, good.
[00:28:54.193]Yeah, Anit.
[00:28:55.026]When you mix atrazine with glufosinate,
[00:28:57.620]so why the ROS goes down?
[00:29:00.942]Okay, so Anit ask why
[00:29:03.790]when I mix atrazine with glufosinate,
[00:29:07.210]why would the ROS go down?
[00:29:09.820]So what we know from chemistry
[00:29:12.480]is the way reactive oxygen species are formed
[00:29:14.830]is then when you have excess electrons
[00:29:17.410]reacting with oxygen, right?
[00:29:21.090]So you have to have electron to form ROS,
[00:29:24.240]they don't form outside of that.
[00:29:26.330]So like all the herbicide that causes ROSes
[00:29:30.630]they somehow either block photosynthesis.
[00:29:34.410]So for example,
[00:29:35.793]so let's start with atrazine, atrazine alone,
[00:29:38.640]we block the flow of electron.
[00:29:41.170]So you would think, well you can have any ROS
[00:29:43.310]but what happens is when we block the flow of electron
[00:29:47.300]at the phytochrome binding site,
[00:29:52.060]the whole photosynthetic apparatus
[00:29:54.780]is designed to capture light energy.
[00:29:58.440]It energizes the chlorophylls, right?
[00:30:00.980]So they get energized.
[00:30:02.360]And then as they go back down,
[00:30:04.330]you have what's called energy transduction
[00:30:06.720]when one electron from the chlorophyll leaves
[00:30:09.370]to go into photosystem two.
[00:30:11.640]Well, that can't happen,
[00:30:12.710]so that extra electron ends up in the oxygen.
[00:30:15.060]So that electron will come from the chlorophyll itself
[00:30:18.640]at that time.
[00:30:19.740]So
[00:30:22.330]what we did is we use low dose of atrazine.
[00:30:28.940]So we didn't wanna use like the plant...
[00:30:31.380]And I had more pictures but I don't wanna show all this.
[00:30:33.720]I had plants where I had the control,
[00:30:36.280]the glufosinate that had about 40% injury,
[00:30:39.100]the dose of atrazine, the plant was green like the control,
[00:30:41.830]no effect at all, but it was enough to slow down
[00:30:45.120]the rate of electron transport.
[00:30:47.240]So now using this as a tool,
[00:30:50.030]no, I'm not saying using this as a way of mixing herbicides,
[00:30:53.920]but as a tool in the lab to study the process,
[00:30:56.650]I was able to show
[00:30:58.320]that if you slow down the flow of electrons
[00:31:00.790]from photosynthesis,
[00:31:05.030]you also with atrazine prevent oxygen evolution.
[00:31:08.530]So then you also have lower oxygen in the plant,
[00:31:11.280]then you don't get the ROS.
[00:31:12.960]So then we knew that the source of the ROS
[00:31:16.840]come from photosynthesis,
[00:31:18.750]even though the inhibition is way down
[00:31:20.830]at the end of photorespiration,
[00:31:24.120]the way it works is you inhibit glutamine sythetase
[00:31:27.360]and the series of events that's very complicated,
[00:31:30.530]blocks RuBisCO, blocks photosynthesis
[00:31:33.880]and then you have these elect, sorry.
[00:31:39.280]Because you have electron transport,
[00:31:41.940]those escape don't have nowhere to go
[00:31:44.270]and they actually then form those reactive oxygen species.
[00:31:47.600]So my point is,
[00:31:50.700]because we get ROS with glufosinate
[00:31:53.360]and we don't have ROS with low rate of glufosinate...
[00:31:56.120]I mean glufosinate with low level of atrazine
[00:31:58.120]that shows that if you slow down oxygen evolution
[00:32:00.360]and electrons, then you make glufosinate not work anymore.
[00:32:06.250]Instead of atrazine,
[00:32:07.366]if you had for example topramezone.
[00:32:10.530]So if you try to use-
[00:32:12.472]Will that have a lethal effect on ROS?
[00:32:14.799]So like the issue of HPPD inhibitors?
[00:32:16.690]Yeah, so that's some of the things
[00:32:18.636]that we're actually, we've not done that yet.
[00:32:21.130]So topramezone...
[00:32:22.250]Anit ask what would happen if we put topramezone
[00:32:24.700]or some kind of other HPPD inhibitors.
[00:32:27.000]So HPPD inhibitors, what they do
[00:32:28.880]is they prevent carotinoids biosynthesis
[00:32:32.870]and carotinoids are required for the stabilization
[00:32:35.950]of the chlorophylls and the photosynthetic apparatus, right?
[00:32:40.180]So in a plant that's treated with HPPD inhibitors
[00:32:46.213]it turn white.
[00:32:47.510]So essentially they have no more electron transport.
[00:32:50.330]So that would be actually interesting to see
[00:32:52.800]if you have a plant that's already white,
[00:32:54.970]if you put glufosinate, will only the green parts die
[00:32:58.740]and the one that are white not burned down?
[00:33:01.820]That would be another good way of testing those hypotheses.
[00:33:05.080]So I would think that
[00:33:07.140]once you destroy the chlorophyll apparatus,
[00:33:09.590]then glufosinate will not have its effect either on it.
[00:33:15.200]Any other questions?
[00:33:16.470]Okay, good, I'm gonna move on to the last part
[00:33:19.650]and then we'll have time for questions.
[00:33:20.760]So this is a synergy between the fashion and PPO inhibitors.
[00:33:25.330]And these actually has resulted with 14 patents.
[00:33:30.690]So it looks great with my boss, my department head
[00:33:32.850]because I have 14 patents,
[00:33:34.620]but actually it's 14 times the same patent.
[00:33:37.970]Essentially BSF made a pattern for every PPO inhibitors
[00:33:41.700]that are commercialized.
[00:33:42.780]So it's the same patent, but one is with lactoferrin,
[00:33:45.440]one is with sulfentrazone, one is with saflufenacil.
[00:33:48.907]But nonetheless, this is actually a very interesting thing
[00:33:51.540]because I mentioned
[00:33:55.400]that when we inhibit glutamine sythetase
[00:33:58.040]you will expect glutamine levels to go down
[00:34:00.780]because you inhibit the biosynthesis
[00:34:02.810]and you would think that glutamate would accumulate,
[00:34:05.860]but it did not accumulate.
[00:34:08.310]So it is like what's going on?
[00:34:09.730]Where's glutamate going?
[00:34:11.570]So because of my background in biochemistry
[00:34:16.170]and my work with PPO inhibitors,
[00:34:19.680]I know that one of the major role of glutamate
[00:34:25.460]is for chlorophyll biosynthesis,
[00:34:28.260]and only people that really study this know this,
[00:34:31.050]but every chlorophyll molecule has eight glutamate in it.
[00:34:35.810]The entire tetra-power ring
[00:34:38.400]is made out of eight glutamates, all right?
[00:34:40.890]So chlorophyll is a huge sink of glutamate.
[00:34:45.920]So again, what we show here
[00:34:50.970]is the untreated amount of glutamine is gray
[00:34:57.480]and then the amount of glutamine with glufosinate,
[00:35:01.290]when you put glufosinate you completely inhibit
[00:35:04.070]the synthesis, you have no glutamine.
[00:35:05.820]However, I wouldn't have expected glutamate
[00:35:07.650]to go up like this, but glutamate goes down too,
[00:35:12.280]which is really not...
[00:35:13.560]If you stop the water out of a dam,
[00:35:15.520]you would think the water would accumulate before the dam,
[00:35:18.230]but here the water does not accumulate,
[00:35:19.750]it goes some other place, right?
[00:35:21.510]Well, I'm tilting my hand a little bit,
[00:35:24.580]but proline is derived from glutamate.
[00:35:27.400]So you can see proline levels go up
[00:35:29.880]and arginine is derived from glutamate,
[00:35:32.170]and that goes up as well.
[00:35:33.270]So that also is a sink for glutamate,
[00:35:36.540]but the most interesting part will be the next thing.
[00:35:38.780]So essentially glutamine decreased, glutamate decreased,
[00:35:42.840]and then proline and arginine increased.
[00:35:46.210]Now that was really strange, so I told my student,
[00:35:49.280]okay, instead of doing a 24 hour experiment,
[00:35:52.440]let's do a time course.
[00:35:54.260]And actually what we see here,
[00:35:56.000]the control, that's the amount of glutamate over controls,
[00:35:58.450]so it stays about the same.
[00:36:00.010]When we put glufosinate within three or two hours,
[00:36:04.110]we got a big spike of glutamate.
[00:36:06.060]So it dials back up above the dam,
[00:36:08.810]but then it's rerouted, it completely reshuffles
[00:36:12.270]or rearranges the fate of glutamate,
[00:36:14.650]it goes away somewhere down to zero.
[00:36:17.620]So this one shows you while some of it goes to proline,
[00:36:21.180]some of it goes to arginine
[00:36:23.280]and I already told you that glutamate also
[00:36:28.090]is involved in chlorophyll biosynthesis.
[00:36:31.490]So one glutamate goes into glutamate semialdehyde,
[00:36:34.670]one of those mix one aminolevulinic acid.
[00:36:39.620]And then, sorry, two of those are necessary
[00:36:42.730]to make one of those,
[00:36:44.480]and then that's converted into porphobilinogen
[00:36:47.290]and it takes four of those to make this,
[00:36:49.700]and then you end up with chlorophyll over here.
[00:36:52.880]And this is why I got excited because I started thinking,
[00:36:55.160]well, if in the presence of glufosinate,
[00:37:00.500]glutamate is shunted to a chlorophyll biosynthesis,
[00:37:04.270]we know that PPO inhibitors act by blocking that pathway.
[00:37:08.740]So I was thinking,
[00:37:09.573]well, what are the odds working on glufosinate
[00:37:12.740]to go back to what I was working on on a PhD, smaller world,
[00:37:16.780]how things kinda work together.
[00:37:17.880]And we know PPO inhibitors
[00:37:19.860]what they do is they cause porphyrin accumulation
[00:37:22.240]and that then reacts with light
[00:37:23.990]and you end up with ROS, right?
[00:37:26.930]So that's another way that you can get ROS in plants.
[00:37:29.457]But it turns out with glufosinate, not only we do that,
[00:37:33.720]we get tons of more porphyrin accumulation,
[00:37:37.850]and I'll show you that in a minute.
[00:37:39.460]So here's an example, for example, this is control plant.
[00:37:43.550]We use half the weight of glufosinate
[00:37:45.650]because we did not wanna kill the plants again.
[00:37:48.500]And this is half the weight of glufosinate with
[00:37:54.540]one-twentieth of the weight of saflufenacil.
[00:37:58.590]So one-twentieth of the regular rate
[00:38:01.460]and we completely smoked the plant.
[00:38:03.240]So essentially a small amount of glufosinate does that.
[00:38:06.250]You put a dash of saflufenacil,
[00:38:09.130]you completely kill the plant.
[00:38:11.140]And that has to do with this,
[00:38:14.310]control of no ROS, glufosinate the low rate, some ROS.
[00:38:18.460]That rate of saflufenacil, the one
[00:38:20.460]that's a small amount of ROS,
[00:38:22.437]put the two together, you get a huge amount of ROS.
[00:38:25.580]And why is that?
[00:38:26.940]Is that the mixture causes a huge amount of protoporphyrin.
[00:38:30.970]So again, understanding biochemistry,
[00:38:33.390]and that's my sales speech for the kind of work that I do
[00:38:36.970]is that I'm a weed scientist,
[00:38:39.340]but I don't think necessarily like a weed scientist.
[00:38:42.740]I think a weed science in terms of biochemistry,
[00:38:45.350]which is really exciting
[00:38:46.280]because thinking about what we know about plants,
[00:38:49.790]we were able to discover there's actually a connection
[00:38:52.920]with glufosinate and PPO herbicides
[00:38:56.380]that glufosinate causes a backup of glutamate,
[00:39:01.100]plant has to do something with it.
[00:39:02.800]It shuns it into chlorophyll,
[00:39:04.920]so that then if you put PPO inhibitors,
[00:39:08.940]you get a huge amount of ROS
[00:39:12.690]and it's not additive, is synergistic,
[00:39:15.350]we've done a whole isobel study.
[00:39:17.740]I don't wanna talk too much about isobel studies,
[00:39:19.510]but essentially additive compounds will be on the red lines,
[00:39:23.770]antagonistic compounds will be above the red line
[00:39:26.700]and then synergistic compounds will be below the red line.
[00:39:29.210]You can see some very strong synergy
[00:39:31.480]between PPO and glufosinate.
[00:39:35.550]So that was really exciting.
[00:39:37.570]That's experiments in the field
[00:39:39.020]where we have low rates of these PPO inhibitors alone.
[00:39:48.180]And if we mix these PPO low rates with glufosinate,
[00:39:51.770]we get a lot more damage in the field,
[00:39:54.030]so it washing the field as well.
[00:39:57.050]And then this is really,
[00:39:58.270]I think one of the most interesting part,
[00:40:01.360]and we're still working on this is that mentioned
[00:40:04.530]that glufosinate sometimes work really well
[00:40:06.560]and sometimes it fails,
[00:40:08.310]and that's because glufosinate is very sensitive
[00:40:10.510]to the environmental conditions.
[00:40:12.480]It is known that it works best at high temperature
[00:40:15.780]and high humidity and in drier environment,
[00:40:19.000]low temperatures is not gonna work at all.
[00:40:21.460]And so what we show here is at 25 degrees for Colorado,
[00:40:27.510]that's in growth chamber, high relative humidity,
[00:40:30.110]but that's about as much as we can get in Colorado,
[00:40:32.570]in a growth chamber.
[00:40:33.640]This is basically glufosinate alone,
[00:40:37.060]the low rate of saflufenacil, nothing
[00:40:39.640]but the two together, it performs, it's dead,
[00:40:43.510]so it's not gonna be more dead than dead, right?
[00:40:45.720]But what we know is that at low temperature
[00:40:50.120]or low relative humidity, we get something like this,
[00:40:53.350]glufosinate does not work really well.
[00:40:55.800]Against saflufenacil, it doesn't do much at that low rate,
[00:40:58.830]but we'll put the glufosinate
[00:41:00.741]and a low rate of saflufenacil
[00:41:02.800]and now we recover the same activity.
[00:41:05.420]So it kind of overcomes the environmental limitations
[00:41:08.040]of glufosinate.
[00:41:08.873]So one of our goals in the whole study was
[00:41:11.970]can we make glufosinate work more reliably?
[00:41:17.193]Now we have greenhouse data that says it does,
[00:41:19.630]the question is,
[00:41:20.750]can we actually make that happen in the field?
[00:41:24.070]And that's why we have those patterns,
[00:41:25.043]because I think BSF is thinking maybe it will work,
[00:41:28.490]they wanted to protect that technology.
[00:41:31.240]I'll stop and have questions.
[00:41:32.840]Anit, yeah, yeah.
[00:41:34.713]So like saflufenacil in a mixture of glufosinate
[00:41:39.104]was more effective than when you compare with mixing of
[00:41:42.576](indistinct)
[00:41:46.549]Yeah, it will be all the same.
[00:41:48.390]It's all the same, they would work all the same.
[00:41:50.100]We used saflufenacil
[00:41:51.420]because by then we were funded by BSF,
[00:41:54.300]so we wanted to show-
[00:41:56.170]In one of the slides, if you can go back.
[00:41:58.550]Oh, in the field?
[00:42:00.740]Yeah, yeah.
[00:42:01.720]Okay, well, flumi is a pre, right?
[00:42:03.660]So to do-
[00:42:06.831]Not flumi,
[00:42:07.664]I was talking more about those lactofen and fomesafen.
[00:42:11.240]Well, I mean, then yeah.
[00:42:12.073]What I'm saying is it's not as good as Saflu
[00:42:15.150]but it's definitely better than this.
[00:42:19.930]So like, if you compare fomesafen alone
[00:42:22.460]and fomesafen with blue, is still a much better.
[00:42:26.395]This I think it has to do maybe with dosages
[00:42:29.730]because what we picked one value,
[00:42:32.290]which was one-twentieth of the field rate, right?
[00:42:36.970]We had to come up with a value
[00:42:39.000]and it could be that if this was not one-twentieth,
[00:42:41.410]but one-fiftieth of a field rate,
[00:42:44.610]maybe you would end up with something like this.
[00:42:46.550]Thus I think this is where all the development,
[00:42:48.990]I think people would have to do field work
[00:42:50.910]to figure out actually
[00:42:51.890]what's the best glufosinate to PPO ratio,
[00:42:56.730]'cause it may not be the same for all of them.
[00:43:00.375]So with those herbicides, like one means?
[00:43:02.390]So normally I think saflufenacil
[00:43:05.920]is only like 20 grams per acre, per hectare, 20 grams,
[00:43:10.330]so it's one-twentieth.
[00:43:12.240]So whatever they field rate
[00:43:13.970]that's one-twentieth of their label field rate.
[00:43:18.100]That's what we have different values
[00:43:19.360]because they're not all the same.
[00:43:21.960]So the value is one-twentieth,
[00:43:23.890]the field rate of this one-twentieth of that and so on.
[00:43:27.550]So what is it that mix,
[00:43:29.650]like we've done some work way back, but not on this level
[00:43:33.590]because none of us here are biochemists.
[00:43:37.750]But when we mix butafenacil with litocin
[00:43:40.820]we're seeing better in back
[00:43:43.530]than it was explained to us
[00:43:45.120]that kind of like litocin
[00:43:46.310]will piggy back some penicillin going down
[00:43:50.340]throughout the plant that's marked in green.
[00:43:53.020]For there, within this case here,
[00:43:57.930]is there any translocation of those two together,
[00:44:01.670]there's one, obvious there's (indistinct)
[00:44:05.200]but what is the trick in that?
[00:44:07.190]You know what I'm trying to say?
[00:44:08.530]Yeah, so the trick here is that glufosinate
[00:44:13.200]makes the PPO chemistry harder
[00:44:16.310]because a rate like this of let's say saflufenacil
[00:44:21.720]is not high enough to cause porphyrin accumulation.
[00:44:25.730]If you flip the two,
[00:44:26.821]if you have both glufosinate at one
[00:44:29.127]and saflufenacil at 20.
[00:44:31.690]Yeah, and so because we had
[00:44:33.770]all these conversations, right?
[00:44:37.110]What we know is that the PPO chemistry
[00:44:41.630]is not helping glufosinate,
[00:44:43.297]and the way we know that is we had Liberty.
[00:44:48.150]Sorry, let me back up.
[00:44:49.030]So yes, we have...
[00:44:52.630]No, sorry, remember this.
[00:44:58.190]We did two things.
[00:45:00.100]One, we did the same experiment or Liberty Link soybean,
[00:45:04.850]and that glufosinate does not cause an injury
[00:45:09.970]to Liberty Link soybean, right?
[00:45:12.230]And if you use a low dose of saflufenacil is not gonna kill,
[00:45:17.440]soybeans is tend to be tolerant to saflufenacil, right?
[00:45:20.233]That's why we can use it on.
[00:45:22.540]But if you mix the two,
[00:45:24.050]it makes it sensitive to saflufenacil.
[00:45:26.710]So it makes the PPO chemistry harder.
[00:45:32.013]It does not overcome glufosinate resistance.
[00:45:34.070]We did the other way around
[00:45:35.940]where we had PPO resistant Palmer
[00:45:42.120]and this does not help,
[00:45:44.530]it doesn't overcome the resistance to PPO resistance, right?
[00:45:48.870]So it goes one way and it doesn't go the other way.
[00:45:52.400]So really it's definitely glufosinate
[00:45:55.137]makes the PPO chemistry harder,
[00:45:57.778]and if your plant is already resistant
[00:45:59.350]and you don't get in porphyrin accumulation,
[00:46:01.300]then it's not gonna cause any porphyrin accumulation
[00:46:02.553]and you're not gonna have any injury.
[00:46:05.030]If you have Liberty Link, which is glufosinate resistant,
[00:46:09.830]you get more PPO injury when you put glufosinate
[00:46:13.500]because you push
[00:46:17.980]some of the glutamate into the porphyrin pathway.
[00:46:20.560]So it's interesting.
[00:46:23.940]The chemistry makes the PPO chemistry harder,
[00:46:26.800]but if your plant is resistant to PPO
[00:46:28.576]it doesn't overcome resistance.
[00:46:31.849]Back to your second part of the mode of action,
[00:46:35.086]all these new findings, do they change anything
[00:46:38.731]about the way you should use Liberty.
[00:46:41.059]Like what are some of the practical implications or?
[00:46:43.503]So the question is whether some of our discovery
[00:46:48.901]have impacted how we use the chemistry.
[00:46:51.650]I think not at this point,
[00:46:53.330]I think they're thinking about it.
[00:46:54.690]I'm sure they're doing a lot more work
[00:46:56.030]and I'm hoping that it will have some impact,
[00:46:58.270]especially if there was a way to use those mixtures
[00:47:02.100]to overcome situations
[00:47:06.240]where or Liberty fails, right?
[00:47:08.430]When the glufosinate does not work well in dry environment,
[00:47:12.210]there could be some really interesting new mixtures
[00:47:14.790]that would extend the usability of glufosinate
[00:47:17.500]into areas that normally is not used.
[00:47:20.622]So it will have an impact?
[00:47:22.701]Yes, right.
[00:47:25.310]It may not pan out,
[00:47:27.180]but at least from a biochemistry standpoint,
[00:47:29.960]it would be a basis why it would work.
[00:47:35.180]Oh, sorry, yes, in the back, yeah.
[00:47:36.797]Did you try out a combination of glufosinate
[00:47:39.667]in PPO inhibitors in different lighting, like settings?
[00:47:43.850]So no, no, we didn't try that.
[00:47:47.250]So the question was, did we try these combinations
[00:47:49.240]under different light settings?
[00:47:52.550]The one thing,
[00:47:53.530]the reason why we didn't categorize that as a high priority
[00:47:56.250]is because we know the ROS are light dependent.
[00:48:00.780]So I don't think this would help
[00:48:02.380]because both the PPO chemistry is light dependent
[00:48:05.260]and glufosinate is light dependent.
[00:48:06.910]So I think that would not address that issue
[00:48:10.750]that you have to have light for both of these chemistries
[00:48:13.610]to be active.
[00:48:15.240]In the back I saw you, yeah.
[00:48:17.270]So when we are talking about glufosinate
[00:48:19.019]in chemical L and D/L phosphinothricin,
[00:48:22.480]why we are considering D as an inactive,
[00:48:24.680]was the part of that molecule or are they similar?
[00:48:28.500]Yeah, yeah, yeah, so I'm gonna do this real quick.
[00:48:31.760]So the question that has to do with a D and D/L glufosinate
[00:48:35.370]and it'll be easier if I do it like this
[00:48:37.890]'cause I can't go back to the beginning.
[00:48:39.680]The difference, they are isomers.
[00:48:43.250]Where it is?
[00:48:44.083]Right here.
[00:48:45.060]So the only difference between a D and D/L
[00:48:50.070]is that this amine group points up on the L
[00:48:54.350]and this amine group points down on the D.
[00:48:58.340]Now one is herbicidal,
[00:49:02.220]the other one is not.
[00:49:03.860]And that has to do, so keep that in mind, right?
[00:49:07.360]That has to do, let me see if I can go to the slide
[00:49:11.500]on the mode of action.
[00:49:12.580]Sorry, I'm going right here.
[00:49:15.660]I show you that the substrate of the enzyme is L glutamate,
[00:49:22.960]all amino acids in biological systems are the L form,
[00:49:27.120]there's no D amino acids in biological systems.
[00:49:30.890]So the enzyme has evolved glutamine synthetase
[00:49:34.400]to use L glutamate.
[00:49:36.770]And the pocket is such that it has a pocket
[00:49:39.250]for the L, the nitrogen sticking up like this,
[00:49:42.350]which is the same on glufosinate.
[00:49:45.330]If you put D glutamate,
[00:49:49.940]this enzyme will not recognize this as a substrate,
[00:49:52.830]and it will not be inhibited by the D form of that enzyme.
[00:49:56.883]That answers your question? Yep.
[00:49:58.780]So, and then the question is
[00:50:00.230]why would companies make both?
[00:50:02.391]And that's because of the cost of synthesis,
[00:50:06.170]two is very difficult to make stereo specific molecules
[00:50:10.800]at an industrial level.
[00:50:13.380]When you do the synthesis,
[00:50:14.720]you end up with a similar mixture of D and L,
[00:50:17.730]and it's a lot cheaper to sell a pound of that.
[00:50:23.420]Only 50% of it is active and 50% is not active,
[00:50:26.680]it's cheaper do that, then actually spending the money
[00:50:29.380]to separate the two.
[00:50:31.420]But they are companies
[00:50:33.700]and there's actually a startup company
[00:50:35.540]that has developed a system to only make D/L glufosinate
[00:50:42.230]so that you if spray half the amount
[00:50:44.180]and get the same effect in nature.
[00:50:46.503]With recreation of some of these
[00:50:48.450]to potentially glufosinate resistant weeds?
[00:50:53.562]So Stevan was asking about what's the...
[00:50:58.040]Do we have a glufosinate that is resistant
[00:51:00.314]or a confirmed or its a lot of-
[00:51:03.100]So Kyle Bruno who was in Oregon
[00:51:06.660]has a population that was resistant to glufosinate.
[00:51:10.790]On ryegrass.
[00:51:12.230]But we did all the biochemistry
[00:51:13.140]and it's not a target site,
[00:51:14.790]it's more of a metabolism based resistance.
[00:51:17.270]Even though they have a mutation that is inherited
[00:51:21.330]on the enzyme, but I tested the enzyme
[00:51:22.640]and it's not resistant.
[00:51:24.410]Now I'm reviewing a paper at the moment
[00:51:27.690]of a resistant target site.
[00:51:31.144]Are they in the country,
[00:51:32.440]glufosinate resistant Palmer?
[00:51:34.440]But not target though, is it?
[00:51:35.837]I don't know the cycle-
[00:51:37.490]Yeah, but I think I'm reading a paper right now
[00:51:39.360]that's pretty convincing but I think they have a mutation
[00:51:41.500]with target site resistance now.
[00:51:44.830]But the reason why it's slow to evolve
[00:51:47.230]is very similar to probably why it took a long time
[00:51:50.400]for glyphosate,
[00:51:52.150]because we're targeting a reaction in immediate.
[00:51:55.040]So it's really difficult for an enzyme to accept a mutation
[00:51:57.850]that provide resistance without really messing up
[00:52:00.250]the biochemistry of the enzyme,
[00:52:02.340]but I think over time, everything will happen.
[00:52:06.140]So it happened with glyphosate,
[00:52:08.760]the more we use glufosinate,
[00:52:10.150]the more it's gonna happen for sure.
[00:52:13.120]Oh yeah, Anit.
[00:52:13.953]So why glufosinate has been
[00:52:15.920]in the market for many years,
[00:52:17.660]but just in the last couple of years,
[00:52:20.003]there's one product came to the market,
[00:52:22.730]which was like three weeks with glufosinate records,
[00:52:24.894]like it was like topramezone.
[00:52:26.934]It was three weeks and yet glufosinate.
[00:52:28.607](indistinct)
[00:52:31.153]Okay.
[00:52:33.030]So there's definitely like compatibility issue
[00:52:35.950]with other herbicides or?
[00:52:37.420]I don't know.
[00:52:38.290]So the question is there's a new product, topramezone
[00:52:42.948]and glufosinate mixed together.
[00:52:45.710]But why has there not be more before?
[00:52:48.560]Yeah, I don't know the answer to that question.
[00:52:50.120]It could be that they are not compatible.
[00:52:52.800]I don't know the answer to that question.
[00:52:54.090]If there's more questions then we'll report,
[00:52:56.302]though really this is great, Franck.
[00:52:58.180]Thank you, thank you.
[00:53:00.156](audience applauds)

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New Insight in the Mode of Action of Glufosinate

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