In this webinar, Dr. John Gammon gives an introduction to PRI and what it can tell researchers about xanthophyll cycle activity, carotenoid: chlorophyll pigment ratios, light-use efficiency, and plant stress. He also discusses remote sensing of PRI.

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Presenter

Dr. John Gammon, University of Alberta

Transcript

JOHN GAMON 0:10
Hi I am here to talk about the photochemical reflectance index or PRI. And this is a story of plant pigments. And this is a picture of the boreal forest in the fall, which is the time of year when we notice plant pigments. And the talk today will be starting with an introduction to the photochemical reflectance index, and it will cover what can PRI tell us. It can tell us about the xanthophyll cycle. It can tell us about relative amounts of carotenoid and chlorophyll pigments. And it can tell us about photosynthetic activity and plant stress under a variety of conditions. So I’ll give examples of those. And I want to end with the theme of remote sensing of PRI.

JOHN GAMON 0:55
So, when you take a walk in the woods in the fall, you often see gorgeous colors. You see the greens of the trees, the evergreens, and the yellow carotenoid pigments, and there’s a bit of red anthocyanins in this. Normally, we don’t notice these different pigments because they’re masked by green chlorophyll pigments. But these pigments vary diurnally, seasonally, and this is what we’re picking up with PRI. So if you look at the actual leaves in the fall, you can see the disappearance of the chlorophyll, and you do notice the carotenoids that have been masked by the chlorophyll. And seasonal changes and diurnal changes in the carotenoid pigment pools are largely what PRI is going to be sensing. So this picture shows you the pigments in a different way, the yellow carotenoids, the red anthocyanins, and the green chlorophyll pigments in liquid amber leafs. And you see the spectra of the three leaves in the bottom, and you can see they have rather different spectra. So what we’re using is spectral reflectance to detect differences in activity and amounts of these pigments. So the groups of pigments I’ll be talking about are primarily— initially the xanthophylls, a group of carotenoid pigments, and they’re a type of carotenoid pigments along with many others. And there’s the chlorophylls as well and the anthocyanins. I won’t be talking about anthocyanins, but mainly the xanthophylls and the carotenoids. And the relative amounts of carotenoid and chlorophyll pigments is the focus of this talk.

JOHN GAMON 2:42
So a little bit of plant physiology, what happens when light hits a leaf? Well, normally in a healthy leaf, it will drive photosynthesis. Chlorophyll or carotenoid pigments will absorb the light. And if it’s the carotenoids, they’ll pass it on to chlorophyll. And that will drive electron transport and normal photosynthesis or carboxylation, the uptake of CO2 and the fixation of carbon dioxide and forming carbohydrates, which is what’s shown on the right of this diagram. That’s what we would consider normal photosynthesis. Now, there are many other things that can happen to that radiation. If there’s extra radiation, there’s a spillover mechanism. It’s also a property of a pigment, it can fluoresce and release extra energy as fluorescence. So there’s constantly a fluorescence signal coming off of plants from the chlorophyll fluorescing. If there’s conditions of stress, and photosynthesis slows down, for example, the stomates close, water stress, temperature stress, nutrient stress, and the plant cannot function fully, its photosynthetic system is reduced in its activity. Then the plant has to find a way to get rid of the extra energy if it’s out in high sunlight. And that’s where the xanthophyll cycle that’s shown on the left comes in. And under extra light, the pigment violaxanthin is converted to antheraxanthin and then zeaxanthin. That’s called deepoxidation. And under limiting light, the reverse pattern happens and that’s epoxidation. And that happens on a diurnal basis as the light changes. But if there’s a lot of extra light, this conversion kicks in and instead of passing the energy on to electron transport and driving photosynthesis, the extra energy can go to zeaxanthin from chlorophyll and spill over or be turned into heat. This is a process that safely dissipates the energy and it’s also called non photochemical quenching because we’re quenching the fluorescent signal.

JOHN GAMON 4:53
So the take home message here is that the xanthophyll cycle is intimately involved with the photo protection of chlorophyll under stress. This is related to the chlorophyll fluorescence. It’s another energy spillover mechanism. And all of these xanthophyll cycle pigments are part of a larger carotenoid pigment pool size. So that becomes relevant in this talk as well. We’re looking both at the xanthophyll cycle and the larger pool size of carotenoid pigments relative to chlorophyll under changing conditions. And we can use this as an indicator of photosynthetic activity and stress. So one way to summarize the change in the xanthophyll cycle is through the epoxidation state, which is shown on the top. It’s the content of the violaxanthin, plus half the antheraxanthin divided by the sum of the three xanthophyll cycle pigments. And that formula is up on top. So the oxidation state, or EPS for short, will come up in a subsequent slide.

JOHN GAMON 5:56
So the story of PRI really began with some experiments back about in the late 80s. And a shade removal experiment with sunflower and it was known at the time that the xanthophyll cycle existed. And since these are pigments, it seemed likely that if you could expose a canopy to highlight abruptly, you should be able to see a change in the reflectance spectrum. And just like a waiter, the old trick with a tablecloth, where a waiter pulls a tablecloth out from under under a bunch of plates. The idea here is to shade the canopy with a black cloth, and then suddenly pull that cloth out and then abruptly expose the canopy to full sunlight at midday. So this canopy has been shaded since the night before. It’s in a dark adapted state. And the thing that this picture doesn’t show is while this is going on, we’re looking at it from above with a spectrometer, which is monitoring the reflectance of that canopy. So from the moment that shade cloth is removed, we’re watching that canopy and monitoring the spectral reflectance. So this experiment revealed that there were indeed sudden changes in spectral reflectance, shown in the top panel. And those are shown as a different spectrum in the bottom panel on the left. And you can see over a time period of several minutes, from half a minute to two minutes, 10 minutes to 40 minutes, you can see a fairly large change that’s shown here. And that has to do with the quenching of the chlorophyll fluorescence. That’s the fluorescence signal disappearing. And it’s disappearing because this other mechanism is kicking in. The xanthophyll cycle pigment conversion is starting, and that shows up here where the blue arrow is, at 531 nanometers. And we knew this was the case because we also took leaf samples from this canopy as these measurements were being made and extracted the pigments and compared the actual pigment content and epoxidation state to these reflectance spectra. And what we found was, over the first 10 minutes, a very strong relationship between the epoxidation state of the xanthophyll cycle pigments and this reflectance signal at 531 nanometers. You’ll also see over time that starts to drift. There’s complicating effects of sun angle changes and canopy structure that come into play over longer time periods. But over the short time period, this is a very good measure — this reflectance at 531 nanometers is a very good measure of what’s happening with the xanthophyll cycle. And I’ll come back to this issue of the drift in a moment.

JOHN GAMON 8:51
So we’ve also done experiments with other methods. The problem of the sun moving through the sky and having to deal with the complexities of canopy structure is sometimes an impediment to doing physiological studies. So to pursue this question further, you can take reflectance off of a single leaf and one way to do that is using a light source and a leaf clip and a spectrometer attached to a bifurcated fibre-optic probe. And the picture in the top shows an actual sample being taken, reflectance sample, being taken from a conifer needle. So it’s possible to take a repeatable reflectance sample from something that’s smaller than a millimeter with this method, and it’s very repeatable and you eliminate the problem of background lighting, shadows, and things like that.

JOHN GAMON 9:42
So work like this has revealed similar sorts of responses. This is on a Douglas fir canopy, and you can see in the top panel a very subtle change in reflectance both in the fluorescence region, which is shown in the bottom panel. The dips in the right right hand side is the double dip from fluorescence quenching. And the dip in the left is the change in xanthophyll cycle pigments showing up at 531 nanometers. Now, it’s really hard to see that change in the original spectra. But if you blow it up, that’s a blow up on the right of the green hump in the visible part of the spectrum, and you can see a subtle change in the top panel in the reflectance around 531 nanometers. And when you take a different spectrum, in the bottom panel, you see that clear dip centered at 531 nanometers. And the edge of that feature is at approximately 570 nanometers. And that makes a good reference for this feature. So from this, we developed the idea of an index, the Photochemical Reflectance Index that expresses that 531 nanometer feature that xanthophyll cycle feature relative to the edge of the feature, a reference. And often 570 nanometers is used, although many other wavelengths have been used in the literature. Now this formula gets used different ways. Sometimes people reverse the formula. But this is one of the more common formulas here, the difference between reflectance at 531 nanometers, and reflectance at 570 nanometers, divided by the sum of the two. So this largely corrects for some of the problems with time and sunlight changing and other background effects. The idea here is to try and zero in on the actual xanthophyll cycle feature with this formula. That’s the derivation of this formula.

JOHN GAMON 11:43
So this formula has been used in a lot of different experiments over the years. And, for example, in this experiment, we’re combining reflectance measurements with fluorescence measurements in a gas exchange system, simultaneously measuring the reflectance and the fluorescence as we change the light and the CO2 concentration and measure photosynthesis. So the top panel shows as we go up in light and down in light, sort of a simulated day and as we stress the plant with a low CO2 treatment in the middle of the day, sort of like mimicking a plant’s stomates closing. In the bottom panel, what we see is the response of PRI to those changes, and we see a step change every time we change the light or when we drop the CO2. And the interesting thing here is to note that it’s almost in parallel with the chlorophyll fluorescence. This is the fluorescence yield, and we see this close interaction between these two methods of dissipating extra energy, fluorescence and PRI. The kinetics are slightly different, but they both represent ways of spilling over or getting rid of that extra energy as heat.

JOHN GAMON 12:56
So from these experiments, we’ve learned that there’s a very close relationship between the PRI and the chlorophyll fluorescence yield. In the left hand panel and in the right panel, we see a close relationship between PRI and actual photosynthetic radiation use efficiency or light use efficiency, often abbreviated LUE, which is expressed different ways, either the photosynthetic rate normalized by incident light or normalized by absorbed light is perhaps a better way to do that. People do it both ways. So the promise here is that this index, PRI, can tell us not only about what’s happening with the regulatory processes of photosynthesis over a short timescale, but perhaps can tell us something about the actual activity of photosynthesis, the uptake of carbon dioxide. So that’s often what we really want to know. So another way to think of this is if you look at a photosynthetic light response curve, this is a typical light response curve of a healthy leaf, and you see the photosynthetic rate plotted as a function of the light intensity. If you look at that point there at low light, you get a certain slope from the origin and at highlight, you get a different slope. That difference in slope tells us how much excess energy there is. And that’s the amount of energy that the leaf has to get rid of, and that’s going to show up in the fluorescence and the PRI signals. This part here, this difference, is a change in the light use efficiency. So as we increase the light, diurnally or through a canopy, we should see a change in the PRI signal. When a leaf gets further stressed, the photosynthetic rate is reduced, as shown in the red curve. But the efficiency is also reduced and that we can pick up as PRI as well. This reduced slope on the light use efficiency curve.

JOHN GAMON 14:56
So, at this point, I’d like to move on to how we can use PRI as a stress indicator under high light or when we add other stresses, for example, in the summer under drought or high temperature stress, or in the winter under cold. These are periods when the plant has more light than it can use for photosynthesis. So it has to invoke some kind of photo protective mechanism. And fluorescence and PRI become important in different ways over different timescales with these different kinds of stresses. So, one question we’ve asked is what happens when you look through a canopy? The top of the canopy gets exposed to highlight but the bottom might be shaded. So this is some work that was done in the tropical forests of Panama using the canopy crane. And we asked the question, how does PRI vary with canopy position from the very top to the bottom? And my colleague here Steve Mulkey is shown in the gondola, riding the gondola from the top to the bottom of the forest canopy. Steve refers to this as getting high and hanging out because you ride up and get high and you have to hang out to hang your instruments out over the canopy to measure the reflectance of the canopy. The results of this study show a very distinct gradient in PRI with light level at the different canopy levels. So the top of the canopy, where there’s high light exposure to the middle of the day, has a very reduced PRI, indicating stress conditions. And as you go down into the canopy, you see much higher PRI levels at the shaded regions of the canopy. Now when we compare this to chlorophyll fluorescence, the fluorescence yield on the top figure, we see a nice correlation between these two measurements again. So we can take these things that were measured in the lab, carry them out to nature, and see the same phenomena through a canopy, for example. And also this changes with time of day. So in the morning, the values tend to be in the upper right hand part of the top figure. And towards midday, the more stress condition, in the top of the canopy, we see a reduced fluorescence yield and we see a reduced PRI value as well.

JOHN GAMON 17:18
So another question is, you know, going to a different biome, in this case, we’re looking at ponderosa pine in the Pacific Northwest, what happens with tree age? Now we know from work of my colleague, Barbara Bond, and others, Mike Ryan, people like that, that as trees get older, they have problems with hydraulic conductance, they have trouble getting the water up to the top of the tree. And in a hot summer day, they may experience drought stress at the top. So we asked the question, can we see a difference in tree age and can we see a difference in water stress using PRI, between an old and a young tree in this kind of a forest? And this figure shows a result of such a test. And you see, this is PRI plotted as a function of PPFD or light during the day, photosynthetic photon flux density, for a young tree and an old tree. And they both start out at about the same point in the morning. There’s a very slight difference in the dark state. But as you go to higher light towards midday, there’s a difference in their responses. The young tree declines a little bit. The older tree, which has a problem with water supply to the top of the tree, shows a much bigger decline in PRI. And this is an indicator of midday drought stress in the older tree. So the difference here in PRI as you go towards the middle of the day picks that up. So this is an example of how you can use a dynamic approach to sampling PRI here, in this case at the needle scale, to pick up differences in the behavior, the physiological behavior of two trees in a forest.

JOHN GAMON 19:01
So, we’ve also done work in the boreal forest, and this shows a picture of the Canadian boreal forest and there’s a picture of leaf reflectance measurements being made with a leaf clip on a conifer in the right hand picture. And we’ve asked the question how much does, when you apply these dynamic approaches to forests, how much does the PRI vary within the canopy under different conditions? So this is a study of the top of the canopy versus leaves in the bottom of the canopy in deep shade for the boreal forests. And the top panel on the left shows you the reflectance of the two leaves, and you see there are differences, and you can see the two wavelengths of PRI there for a sun and shade leaf. And the bottom shows you the difference from the dark state to the light state in reflectance, the delta reflectance. And you see that clear double dip of chlorophyll fluorescence quenching and a very strong signal of the xanthophyll cycle in the case of the sun leaf, and a weaker signal in the case of the shade leaf. This tells us a lot about the capacity of the leaf to respond to sunlight. The shade leaf lives in the shade, it doesn’t have as much of a xanthophyll cycle pigment pool, whereas the sun leaf at the top of the canopy has a much larger pigment pool and is much more responsive to light and it has more of that photo protection built in. And we can see that dynamically in this plot on the right, which is a plot of PRI as a function of exposure time to high light. So starting with a dark adapted leaf, we can see how these leaves, the shade and the sun leaf respectively, in the top and the bottom, change from the dark state to the light state. And both of them change. Both of them exhibit a change in PRI. But the sun leaf has a larger change. And that dynamic part is the xanthophyll cycle operating. And you can think of this as a facultative effect, it’s changing within the timescale of a day. And that’s very dynamic. It changes from minute to minute as the light changes. Now, what we also noticed is this fundamental difference in the starting point between the dark and the light, in other words, the shade leaf and the sun leaf. And that difference shown on the left is really due to different levels of carotenoid pigments and relative to chlorophyll, the so called pool size effects. you can think of this as a constitutive of effect. This changes very slowly, and it’s not going to change within a day, but it might change over the life of the leaf or with seasons or with chronic stress. And so there’s more than one thing going on here, and you can tease them apart based on the kinetics. And you can use these dynamic approaches to see the difference in in the xanthophyll cycle or longer term, constitutive effects, pigment pool size effects, on the other hand. And the take home message from this is that within a canopy, as a boreal forest stand, most of the variation in PRI is actually attributable to these pool size changes, not the subtle diurnal changes. So if you were to just measure PRI at random and look at random variability in that signal on these forests, it’s the carotenoid chlorophyll pool size effects that are really dominating this signal. And buried within that is the subtle diurnal change that is the facultative effect.

JOHN GAMON 22:28
So it’s hard to measure things across time and space, which is what we’re talking about with the measurement of PRI. So this has led to some new techniques for measuring spectral reflectance. And what’s shown here is a robotic cart for measuring reflectance across a transect through time and space. And we call this the tram system. This is a chaparral system in California, and you can see a downward looking fiber optic that is connected to a spectrometer. And also on this cart is a upward looking channel that’s reading the downwelling irradiance the solar spectrum, if you will. And so this allows us to correct the reflected radiation by the downwelling irradiance. So as clouds change, or the sky conditions change, we can correct for that with this kind of a dual channel system, so by measuring up and down at the same time. Also, we have other instruments here, like in this case, a thermometer measuring surface temperature. And here’s a picture of the same landscape before a fire came in and burned it. And you can see the tram operating in the background. And we asked the question, how does PRI vary seasonally in such a landscape? And this remember, is an evergreen chaparral landscape. And the results are shown here in this figure by my colleague, Dan Sims. And you can see a seasonal change in the leaf PRI, which corresponds to what we saw with the whole canopy measurements and the tram. And you see two lines. One is the measurements in the dark state in the morning. The other is measurements at midday, in the light state with the open symbols. And you see a subtle difference from morning to midday. But you see a much bigger difference from the first year to the second year or from season to season, winter to spring to summer, for example. And so the take home message here is that there’s a very strong seasonal change in the PRI and a much more subtle change that’s the diurnal change.

JOHN GAMON 24:34
And we looked at this also — by the way, the second year was a drought year and so you see a much reduced summertime PRI value under drought stress than you do in a more normal year in the left. And in this case, it was quite clear that most of the variation in PRI is attributable to changes in the chlorophyll carotenoid pool sizes — the constitutive effect rather than the diurnal effect. And this has been hard to tease apart because when you compare pigment measurements to the PRI measurements, and this is work of a colleague, Cat Stylinski, we see good correlations between PRI, at least for canopy scale and pigment levels measured as xanthophyll cycle pigments or as carotenoid to chlorophyll ratios. So it’s a little hard to know how to tease this apart, but we can do that by looking at it dynamically through time in the figure on the left. So we also ask the question, What about other kinds of ecosystems that undergo strong seasonal changes? For example, in the boreal forest in the wintertime, there’s lots of evergreens. What happens to these evergreens in the winter? Now most of us probably don’t think about this, because we look at evergreens, and we think of them as being green all the time. But if you look carefully, a careful observer will notice that there’s actually subtle changes in the pigment pools. And I have a little prop here. This is a juniper plant from outside, and in the wintertime, they get a little bit dull green, sort of yellow green, and in the summer, they turn a brighter green. And the question is can PRI pick up on that and can it help us monitor those changes? And it turns out it can. Chris Wong is shown measuring PRI on leaves in the left in the wintertime. And the same leaves in the right, you can see, are quite bright green in the summer. So this is ponderosa pine, and also Pinus Contorta, or two species of pine being measured here. And the left you see the needle clip, the leaf clip, and in the right , you also see automated measurements. In this case, we’re starting to use, in addition to spectrometers, some new sensors from a variety of different sources, including the Decagon SRS sensor, monitoring PRI changes in the canopy through the seasons. And what we see is that in the spring, from winter to spring, we see a clear transition in PRI. And we can see this, whether we’re looking at the leaf scale or at the canopy scale, or whether we’re using automated sensors like the SRS sensor. So regardless of what scale or what method we use, we see this spring activation, this change in pigment pools that happens to be correlated with the turning on photosynthesis in the spring. So in these seasonal studies, what we’re learning is that PRI is a pretty good indicator of seasonal changes of photosynthetic activity in evergreens.

JOHN GAMON 27:38
So to sort of summarize these stress effects, PRI detects the xanthophyll cycle, but it also detects the pigment pool sizes of chlorophyll and carotenoids. And the first one, we could call it the facultative one, and the facultative response that operates diurnally, we’re measuring the epoxidation state of the xanthophyll cycle pigments essentially, as they change with light and conditions during the day. The pigment pool size effect doesn’t change on a diurnal basis, but may change seasonally or with chronic stress. And that is also detectable by PRI. And it’s actually a much bigger signal, and is very easy to detect with this index.

JOHN GAMON 28:20
So what are the implications of this? Well, when you think about evergreens, they cover a huge part of the planet. And for example, the boreal forest, about a third of the world’s forests is boreal forests. And the boreal forest has a lot of conifers, and they’re exposed to extreme conditions. And we now know with PRI, we can detect the seasonal changes in photosynthetic activity, as well as diurnal changes in photosynthetic activity. So the asterisk is to indicate that we can detect these changes at different timescales, looking at different kinds of effects with this one index, PRI. And there’s also hope that if we can scale this up, we can use remote sensing to improve our knowledge of photosynthesis. And this can tell us about phenology of the forest and the carbon flux of the forest. And we believe that that may be changing. So how do we monitor this with remote sensing? Well, one way might be to use PRI. So in the last part of the talk, I want to ask the question, how well does PRI scale if we go from the leaf and the bottom part where we might measure photosynthesis in a gas exchange chamber to a tram where we might measure landscape with eddy covariance or other kinds of remote methods, shown here, aircraft and satellite measurements? Can we measure a coherent signal at all these different scales? And can we relate this to other processes of interest like photosynthesis? And it turns out that as long as you have a closed stand and you have solid mass of vegetation, there’s a pretty good relationship between what we measure at the leaf scale and the stand scale. This is a experiment looking at simultaneous PRI measurements at the leaf level and the stand level. For a stand exposed to shade, the shade is removed, and then the shade is put back. And you can see the responsiveness of this signal at two scales. And if you look at this for many, many different plants, leaf versus stand scale, you do see that as long as the canopy is closed, you can see a coherent signal at a stand scale.

JOHN GAMON 30:31
So I think there is hope that we can use this for remote sensing. And one way to do this is to take the PRI signal and put it into this kind of a model, what’s called the light use efficiency model, where the gross photosynthetic uptake is a function of absorbed light times the efficiency with which the absorbed light is used for photosynthesis. So that absorbed light term or A PAR term is shown on the left. And that’s a product of the photosynthetically active radiation and the fraction of absorbed radiation, the fraction of that radiation absorbed by green plant materials. So that together makes the absorbed light term. And then the term on the right is the efficiency term. Now we can measure the term on the left with vegetation indices such as a normalized difference vegetation index, a greenness index. The term on the right, the question is, can we use PRI, or fluorescence of some kind to measure that non photochemical quenching process and to measure the pigment pool size changes in the case of PRI to look at seasonally changing, diurnally changing light use efficiency. And so there have been a number of measurements made and publications made that look at this. And one of the ones that I think is a great example is work by Carolyn Nichol and colleagues showing you airborne PRI measurements of a boreal forest and relating that for several different canopies to the light use efficiency of those same forests measured with eddy covariance. And there’s a significant relationship between the PRI measured at different dates on these different canopies and the light use efficiency.

JOHN GAMON 32:11
Similarly, a colleague of mine Faiz Rahman did a study in the boreal forest using the avarice sensor, where the PRI is put into a model to measure light use efficiency and combined with NDVI. And that modelled product is compared to the carbon flux measured by eddy covariance of various boreal forest sites. And in this case, we see a significant relationship between this model and the carbon flux. And based on these kinds of approaches, you can actually map photosynthesis in real time, using PRI as kind of a dynamic indicator of how photosynthesis changes in time and space. Now, this needs a lot more testing, but this is a real promising direction to go. And my hope is that in the future, we will be able to routinely use these kinds of approaches to look at forests like this and to actually use the spectral information not just, you know, a simple approach but a much more nuanced approach that takes advantage of this spectral information to tease apart the subtle changes in photosynthetic activity that are happening on different timescales. And if we can put that into the right models and verify those models, I think we’ll have some more powerful tools for applying PRI at a large scale and improve our photos and photosynthetic monitoring of large areas of the planet. Thank you very much.