 At this point, I would like to introduce our first speaker, Don Ortt. Don is the Robert Emerson Professor in Plant Biology and Crop Sciences at the University of Illinois. The title of his talk is Improving Photosynthetic Efficiency as a Carbon Mitigation Strategy. Off to you, Don. Thank you very much, Sarah, and thanks to the organizers for the invitation to speak today. So by way of introduction, I should say that for the last eight to nine years, we've been working on the overall strategy of improving photosynthetic efficiency as a way to improve productivity in crop plants. And I think I interpreted my invitation to speak at this meeting to explore the extent to which improving photosynthetic efficiency might be a carbon mitigation strategy. And that's what I will try to do. And so what I'd like to do in the next 20 or so minutes is I'd just like to review the overall rationale of why we think improving photosynthetic efficiency is a sensible target for improving productivity of food crops. I'd like to give two examples of ways in which we have engineered photosynthesis and have proof of concept that we can substantially increase photosynthetic efficiency that leads to increased biomass production. And then finally, I'd like to see if we can set an upper limit on what improving photosynthetic efficiency in managed crop and pasture systems might be able to do as a carbon uptake and a carbon storage strategy. And so it turns out that at least on an energy basis, the process of photosynthesis that has evolved is not so impressively efficient. And so we're certainly not the first to make this calculation. But if you start out with, say, 100 kilojoules of solar radiation, about half of it is outside the photosynthetic spectrum. And so photosynthesis is driven by visible light, which is essentially the light that we can see that's about 400 to 700 nanometers. Plants are green. The reason for that is some of that light's reflected, and so that's wasted. All of photosynthesis is driven by the energy of red photons. And so the 75% of extra energy in a blue photon is simply wasted and so called here as photochemical inefficiency. And then this calculation bifurcates whether you're talking about a C3 or a C4 plant. And the reason is that in C3 plants, they take three ATPs and two NADPHs to reduce CO2 to the level of carbohydrate. But C4s take five ATPs and two NADPHs to do the same thing. And what the C4 gains by investing that additional amount of energy is they're able to concentrate CO2 around the primary carboxylase rubisco and prevent the oxygenation reaction, which I'm going to talk a little bit more about later on. And therefore, largely suppressed photorespiration, which is an energy-intensive process. And so in this calculation, it just shows photorespiration is zero, but there is certainly some photorespiration in C4 plants. And then finally, there's a deduction for autotrophic respiration. That is the maintenance respiration that plants use to maintain themselves and grow. And so at the end of this calculation, we would say that the maximum efficiency of C3 photosynthesis in the way photosynthesis is evolved is about 4.6%. And for C4 plants, it's a bit better at 6% under current atmospheric conditions. And so the maximum that's been observed for a C3 plant during a growing season is about half of what the maximum is. And the average that's observed for farmers in developed countries is about a third of what the maximum observed efficiency is. Stories pretty much the same for C4 plants. 6% efficient, maximum observed for short periods during a growing season, about half that. And the average is observed is about a third in the maximum. And so there's a number of interesting discussions that you can have here. And so why is the maximum so far below the theoretical for both C3 and C4? Or why is the average so far between below the maximum? And we could also have a conversation or other engineering solutions that could actually increase the theoretical maximum. And so as I said, the reason that we got into this is that we were interested in addressing this problem. And that is the UNFAO and others have forecast in order to meet the agricultural demand at the middle of this century that we'll have to increase demands. We'll have to increase supply somewhere between 17% and 120%. The UNFAO said we needed to double it. And certainly that's happened before. It happened in the Green Revolution during the time between 1960 and 2005. Population increased about 110%, whereas crop productivity increased by a crop production increased by 165%. And it's pretty important to understand that of that 165%, 135% was due to intensification. That is growing more produce on a given land area basis. And only about 25% was recruiting new land under cultivation. And most of that was in Africa. And what this graph shows is that at least when we look at small grains or small grains plus soybean, the total land under cultivation probably peaks somewhere between 2000 and is either plateaued or maybe is slowly declining. And you can see the prediction here as we go toward mid-century that the amount of land per person is going down. And so that would require an increase further intensification and more produce per land area. But what you can see from this graph is that's not actually happening. And so we put this together from UNFAO data. And the way to read this slide is, so for the decade ending in 1977, the per decade increase was about 3% per year. But as you can see for these major three crops, two of the major three and nine out of the 10 most important crops in the world, that increase per year is declining. It's declining for several reasons. But one of the reasons for stagnation is that genetic potential or yield potential is not increasing as it was during the Green Revolution. And so those things that drove the Green Revolution, which were largely maximizing harvest index and intercept efficiency, have largely been maximized. But the third component of the yield potential equation, which is photosynthesis, wasn't improved at all during the Green Revolution. And so this is why we think that if we're going to have another doubling of productivity, that it's going to have to involve increasing photosynthetic productivity. And so we've seen this slide before earlier in this presentation. And it just shows that over the last 500,000 years, the CO2 concentration in the atmosphere is significantly below what it is today, which is about 410 ppm. And an increase from these levels of about 270 ppm up to 410 in about 150 years. And so it's probably not surprising to discover that photosynthesis is really optimized and see three plants to the atmospheric concentration of about 250 parts per million. And so what that means is that as CO2 concentration goes up, the rate of this reaction, this carboxylation reaction that is catalyzed by the single protein rubisco is accelerating. And what we could show for modeling as well as experimentation that what's becoming increasingly limiting as CO2 concentration goes up in the atmosphere is the regeneration of the CO2 acceptor RUBP. And so RUBP regeneration involves lots of different enzymes as well as the production of ATP and NADPH. And so we used a modeling approach to try to discover if there were particular proteins within the regeneration of RUBP that were significant or were more limiting than others. And so what we did was we just asked the question for a given investment of nitrogen, where would you put it to optimize photosynthesis? And so when we did that with this model that involves an evolutionary algorithm, you can see that the enzyme C-dehepulus bisphosphatase, it suggests that as CO2 concentration goes up, that the amount of this enzyme needs to go up. And so in this modeling case, we're using CO2 levels at 2005 versus optimizing photosynthesis for the level that we expect in 2050. And so what we did was to overexpress SBPAs in tobacco plants. And when we did that, and so what you see here, we've grown these plants with SBPAs over expression versus wild type. We've grown them in our face facility at both 400 ppm and 750 ppm. And so these were actually in chambers. This wasn't in the face facility. And what you can see is when you look at the rate of photosynthesis versus the CO2 concentration within the leaf, the plants with the overexpression of SBPAs, which are lines 30, 11, and 60. And so these are three independent transformants. We see a higher capacity for photosynthesis. When we move these plants into our face facility outside, you can see we have the daily integral of photosynthesis. And so that's the photosynthesis that occurs across the day. And if you look at the right side of this panel, you can see that there's about a 15% increase in the daily integral of photosynthesis in the plants in which we have increased the SBPAs activity by a factor of about three. That increase in 15% of the daily integral of photosynthesis translates into an increase in the above ground dry matter biomass of about 20%. And so here's a way that we have rebalanced photosynthesis to account for the elevated CO2 concentration, in this case predicted for 2050. And you can see that by doing that, we've been able to increase photosynthesis and increase biomass accumulation at least in tobacco. And so that was an example of moving the observed photosynthetic efficiency closer to the theoretical. And I want to give you an example, again, in the C3 plant of a way in which we might be able to increase the theoretical efficiency. And I've mentioned this process of photorespiration before, and that arises from the fact that this primary covoxylates Robisco, which evolved at a time when there's no oxygen in the atmosphere. Once photosynthesis started oxygening the atmosphere, Robisco was confronted with the problem that it wasn't able to completely discriminate between CO2 and oxygen. And so in our current atmospheric conditions about every fourth catalytic turnover Robisco is rather than fixing a CO2 molecule, it fixes an oxygen molecule. And this is essentially an anti-photosynthesis. And so when it fixes an oxygen molecule, instead of making the two molecules of phosphoglycerate to make when it fixes the CO2 molecule, it makes one of these phosphoglycerates and one of these two carbon compounds glycolate. The difficulty with glycolate is twofold. One is that because it's too carbon, it can't be put back into the C3 cycle. And secondly, glycolate is actually inhibitory to several of the enzymes in the photosynthetic reduction cycle. So when higher plants were confronted with this, they repurposed the biochemistry that they had at hand and evolution came up with an extremely complicated way in which to recycle glycolate involves four compartments in the cell and it uses a lot of energy. And so it evolves ammonia, it evolves CO2. And so for each oxygenation reaction, it uses three and a half ATPs and two and ATPH equivalents. And so to illustrate what that really means, we took a multi-layer canopy model. We parameterized it for soybean and so this is for a mature soybean canopy. And what this shows is that as a function of time a day, during much of the day, about 40% of the ATP and about 30% of the NIDPH that's produced by photosynthesis is actually used to recycle and recapture the glycolate. And so it's hugely expensive. And so what we saw to do was to install a more energy efficient synthetic pathway into the chloroplast of plants and thereby bypass the native pathway. And so we tried a number of different synthetic pathways and the one that worked as best is shown here. And so what we did was introduced into the chloroplast glycolate dehydrogenase from Clamidomonas to convert glycolate to Glock's late in the chloroplast rather than the peroxisome. And then we introduced maleate synthase to take glycolate to maleate within the chloroplast. And then the chloroplast has the ability then to decarboxylate the maleate. And so we're producing CO2 right in the chloroplast rather than the mitochondria raised in the chloroplast CO2 concentration and thereby lowering the competitive reaction, the competition with oxygen. These data on the left-hand side simply show that we're able to express these genes in the transgenic plants that we were able to target the proteins where we wanted them to go in the chloroplast. And the other thing that we wanted to do was now that we have this more energetically efficient pathway in the chloroplast is to drive as much glycolate flux through it as possible. And so we down-regulated the exporter glycolate out of the chloroplast into the native pathway. And so I said that it's more energy efficient and one way to verify that that's actually happening experimentally is to look at the quantum yield of photosynthesis. That is under low light conditions, how many quantities it take to reduce CO2 to the level of carbohydrate. And so you can see we have our empty type or wild vector, and then we have three independent transformants for the synthetic pathway within without the down-regulation of the glycolate exporter. And what you can see is that in many of these cases and we've done this under 400 ppm CO2 and then under a much greater photorespiratory pressure of 100 ppm, in most of these cases we're seeing as much as a 20 to 25% increase in the efficiency of photosynthesis. And so when we moved this into field studies and so we did a couple of field trials, I mean what I'd like to point out to you in field trials again we see this 20 to 25% improvement of the quantum efficiency photosynthesis and with the synthetic pathway we're seeing 20 to 25% increases in biomass production. And so I've shown you two examples. We have a number of others in which we've been able to increase photosynthetic efficiency and that increase in photosynthetic efficiency has led to greater biomass accumulation under replicated field trials. And so then we, and so my purpose here then was to ask the question, can we try to determine what the upper capacity is this, this is for carbon uptake and storage? And so if we were to take all of the managed crop and pasture land on the globe, and so these are areas where we could arguably put plants that we've developed that have higher photosynthetic efficiency, what would that do in terms of carbon storage? And so these are data that Chris Field published some years ago looking at how much crop and pasture land exist on the globe. And so it's broken down an area, the mean net primary productivity of that and then the total net primary productivity on the globe. And so one of the interesting things that this shows is that if you take the total crop pasture land and the abandoned land, these sum to about a third of total terrestrial net primary productivity on the planet. So these are certainly not small contributors and so there is a large amount of managed land that's out there. If we then do the hypothetical experiment of taking all of the crop plants and all of the pasture plants on those lands and replace them with plants that have 25% higher photosynthetic efficiency, you can see that it increases the net primary productivity by 25%. I mean, because we were trying to get at what the upper maximum might be, we've returned the abandoned land and we've given the same level of productivity of the crop land, which is all certainly not true. And so we get an increase of the net primary productivity of these managed lands of about 25%. And so then how do we think about this then in terms of carbon storage? And so if we use this kind of represented in this slide, the relationship between net primary productivity, net ecosystem productivity, and net biome productivity, and we assume that the medium term carbon storage at NEP and the long terms carbon storage at NBP are what we're really after. Then we see that when we factor in the fact that the pasture and crop land is one third of the total, we see an increase of 3.6 gigatons for NEP, it goes up 0.6, and for NBP, it goes up 1.2. And so certainly it's unrealistic to say that for crop and pasture land that we're gonna have medium term storage that is identical to what we have for all NPP. Because certainly some of that medium term storage are things like tree trunks and so on, which we don't have. In these crops. What this comes down to is that if we used all the managed land, all the crop and pasture land, we could offset 7% of global anthropogenic carbon emissions globally. And certainly this is unrealistically high because certainly we're not going to be able to do this on all lands. And as I already mentioned, this medium term carbon uptake isn't a realistic estimate. For crop plants and pasture plants. And so probably something under 1% is what we could expect. And so I think that what we can say is that this strategy of improving crop productivity and land area basis by engineering more photosynthesis, this turns out to be successful. I think that it's greatest impact on carbon storage that we can hope for, maybe reducing pressure on land conversion from land that already has carbon stores to row crop agriculture that tends to release them. And so I will stop there. Thank you very much. So we have a question. The first one will go to Damien Allen. Damien, would you like to unmute yourself when you're able to and ask your question? Hey, Don. Yeah, the question about what are you simplifications of increasing photosynthesis to the mechanisms that you've worked on? Would they, would they be able to do that? Would they, would they go up, would water use go up in parallel or would it stay the same? It's nice to hear from you, Damien. And so we've measured this for the bypass tobacco plants and it stays the same. And so what complicates it is the fact that you would expect an increase in water use efficiency, but what complicates it is that you're dealing with bigger plants and you're dealing with more evaporative area. And so at least in the one experiment that we've done with these plants, it doesn't, it doesn't change. We grow plants in elevated CO2, then we do see an improvement in water use efficiency, but it's not necessarily as large as you expect because of the fact that the canopy is bigger. Thanks. Thanks, Don. We have another question from Shafiq Jaffer. Shafiq, would you like to ask a question when you unmute yourself? Sure, thanks, Jenny. Don, I had a couple of quick questions perhaps. One is, you know, you state these photosynthetic efficiencies, is that the same in the urban farming in the greenhouses where the light is being tailored to the specific wavelengths? Are you seeing the same sort of photosynthetic efficiency? Photosynthetic efficiency is dependent on light color. And so, you know, for example, if you just illuminated plants with light that is 680 nanometers, it would be on an energy basis, it would be way more efficient than what you get with the full spectrum solar energy because there are a lot of those things that wavelengths outside of the photosynthetically active spectrum or the fact that blue light, you know, has energy that's not used by photosynthesis. And so, you know, and if you do it just on par, it automatically doubles it. And so, yes, if you're going to give artificial light and you're going to spend money to provide that light energy, then certainly doing it in the red region of the spectrum is an advantage. You know, there's photomorphological things that you have to deal with. And so there are other colors of light that need to be there, but certainly the main wavelength-driving photosynthesis, you're best off if it's in the red. And the other question I had was kind of around the broad use of this kind of approach across different crops. I mean, you've tested on a few crops. Do you think this kind of engineering the photosynthetic pathway is generalizable across different crops or do you think it's very specific to every crop that you are now? Well, and so, you know, for instance, you know, bypassing the photorespiratory, the native photorespiratory chain is going to be much more important in C3 crops than C4 crops. You know, having said that, what we've done so far, or at least published so far has been in tobacco. And so, of course, producing better tobacco isn't necessarily what we want to do. And so now we're trying to move these into crop plants and see if this increase in biomass, one, that we see it in other species and then secondly, whether that translates into increased yield. And so this summer, we're doing an experiment with potato and so that's one of the first crops that we've gotten these into. But we have hundreds of independent events at the T1 level in soybean. We're putting it into Calpe and we're putting it into rice. And so, you know, right now the jury's out. You know, we're certainly optimistic, but we don't know. I mean, the question you're asking is absolutely the key question as to whether this is gonna be a successful strategy. Thank you very much, Don. Appreciate it. Thank you. And I think we can hopefully squeeze in one more question. Apologies if I mispronounce your name, Jagabandu Hazra. When you're ready, would you like to unmute yourself and ask your question? Sure. Thank you for the nice presentation. So just I have a quick query. So could you please comment on the correlation between photosynthesis and crop yield or productivity? Is it linear? And so that's a bit of a difficult question. And so if you call photosynthesis daily integral of carbon yield, of carbon gain, then there's a good correlation between yield and photosynthesis. If you look at photosynthesis as the maximum rate of photosynthesis of a leaf at the top of the canopy, then there's a very poor correlation between that rate of photosynthesis and yield. One of the things that I do think is interesting is if you plot yield versus total light absorbed by a canopy across many different kinds of canopy, it's a pretty straight line. And so there is that correlation of absorbed light and total production, even though a lot of that light is wasted due to the fact that in many instances, the top of the canopy is way over saturation. And so if you measure the right component of photosynthesis, yes, there is an association of photosynthetic performance and yield.