In this presentation, Dr. Maria Maldonado outlines the critical role of plant respiration in global carbon balance and agricultural productivity, and describes how technical challenges have limited structural knowledge of plant mitochondrial complexes. To overcome these hurdles, her postdoctoral work introduced a non‑traditional workflow that combines partial biochemical purification of mung‑bean mitochondria with a single cryo‑EM data collection of a heterogeneous sample, followed by computational classification to isolate individual complexes. This approach yielded high‑resolution structures of plant Complex I (including a carbonic anhydrase heterotrimer), Complex III dimer, Complex IV (a ten‑subunit assembly), and a Complex III–IV supercomplex, revealing numerous plant‑specific subunits, novel helices, and unique interfaces. Further, conformational dynamics analyses of Complex III uncovered coordinated dimer movements and iron‑sulphur cluster motions, leading to mechanistic hypotheses about the coupling of respiratory and non‑respiratory functions. The work establishes a structural foundation for engineering more selective agricultural inhibitors, improving crop yields, and guiding future studies of respiration across diverse photosynthetic organisms, with broader implications for climate mitigation and biomaterial production.
Chapters
Thank you. Well, thanks everyone for being here. And thank you for this great opportunity to present my work, the structural characterisation of plant respiratory complexes. So plants are the foundation for human society and in fact, most life on Earth. So through— if you think about it, they provide us with the oxygen that we breathe, they are the basis for all the terrestrial food chains, including all the food chains that Maureen just talked about. Plants also provide us with different materials, fuels, housing, for fuels, housing, clothing, and other essentials for us to survive and for society to thrive. But in order for plants to survive, they must carry out two essential metabolic processes: photosynthesis in the chloroplasts and respiration in mitochondria. And these two metabolic processes provide the plants with the energy and the metabolic intermediates they need to live and grow.. And in fact, the balance between the carbon that is gained through photosynthesis and the carbon that is lost through mitochondria through respiration directly impacts the rate at which the plant can grow and the amount of carbon dioxide that the plant can capture or release. And so our understanding of these two essential metabolic processes has global implications in terms of crop efficiency and atmospheric composition. And our understanding also determines our ability to to engineer and implement new strategies to combat climate change and to improve our ability to feed the growing population. However, although we understand the proteins that carry out photosynthesis in great biochemical detail, our knowledge of the proteins that carry out respiration in plants has lagged much behind. And this is mostly to do with strong technical challenges in obtaining sufficient amounts of plant mitochondria for biochemical and structural work. So this gap is represented here on this slide where you just see, which is in a cartoon form. So we do know that in plants as well as in all other eukaryotic organisms, respiration occurs through the mitochondrial electron transport chain represented here. This chain is composed of 4 large membrane protein complexes: complex I, II, III, and IV, composed of multiple protein subunits. And we also know that these complexes can exist individually or they can come together into higher order assemblies called supercomplexes. However, at the beginning of my postdoc, this was pretty much all we knew about these plant respiratory complexes, and they remained in this cartoon representation or low-resolution representations. But if we want to fully understand plant respiration to be able to, for example, manipulate plant metabolism to produce better crops, then we really need to be able to biochemically and structurally characterise plant respiratory complexes. So this was the goal of my postdoc, to overcome the challenges related to studying mitochondrial respiration in plants, to be able to characterize these proteins. So in order to do this, we developed a new or a non-traditional approach of using a partial biochemical purification followed by in situ purification of the complexes. So we start with mung beans, which is our model organism, and we first isolate the mitochondria from them. And then we extract the membrane complexes with gentle detergents and proceed through an initial partial purification using a sucrose gradient. Now traditionally in structural biology, you would follow this initial partial purification with a full biochemical purification, usually using chromatography, to purify each of the complexes of interest and then use each of the pure samples to proceed to the structural data collection, be it with crystallography or in my case cryo-electromicroscopy or cryo-EM. However, this approach of doing a full biochemical purification would be really prohibitive for the plant respiratory complexes because we have just so little of them in the samples, our samples are so limited. So rather than using this traditional approach, we decided to go with the non-traditional approach of using this partially purified mixed sample as our cryo-EM sample. So we took the fractions from the sucrose gradient and used them as this heterogeneous sample for a single cryo-EM data collection, after which we purified the complexes computationally using cryo-EM classification algorithms. So this in practice is what it looks like. So each of the cryo-EM micrographs contains particles highlighted here in yellow, but these particles are a mix, like I said, of all the respiratory complexes. So then we need to use the computational algorithms to separate the particles into the different subsets corresponding to the different complexes of interest, as you can see represented here. So using this strategy, we were able to obtain high-resolution structures for several respiratory complexes from one single grid. So here, these videos show you the reconstructions and the models for a Complex I assembly intermediate called Complex I*, as well as for the Complex III dimer, as well as Complex IV and the supercomplex assembly between Complex III and Complex IV. And now together, these first high-resolution structures of the plant respiratory complexes allow us to start replacing the cartoon representations with the molecular details. Shown as previously unknown features of plant respiration. So for example, our structures revealed several subunit compositions that were unknown from previous biochemical and genetic and mass spec studies. For example, our structure of Complex I showed us that Complex I's carbonic anhydrase domain, which is present in plants but it's not present in mammals or in yeast, is a heterotrimer. And by looking at the structure, we were able to determine the composition of this heterotrimer and basically find out which 3 out of 5 very, very similar protein homologues are in fact the components of this domain. Similarly, our structure of Complex IV also showed us the number and identity of subunits in plant Complex IV. And again, by looking at the structure, we were able to determine that plant Complex IV has 10 subunits, and this is different from other organisms. And looking at the structure, we were also able to determine that 3 subunits here on the right, which were previously thought to be plant-specific because their sequences are highly divergent versus mammals and yeast, are in fact homologues of the subunits previously seen in the other organisms. Additionally, the structures revealed several plant-specific arrangements, for example, several new helices and beta sheets in Complex III's catalytic matrix domain. As well as a plant-specific interface between Complex III and Complex IV in the supercomplex. So for example, if you compare the regions encircled here in the dashes, you can see that there are very large differences between yeast and plants. And we hypothesise that these differences have functional implications for the plant supercomplex and Complex III activity. Moreover, in addition to using our cryoparticles to to determine the static structures of the complexes, we used a different programme algorithm to examine the movement of the complexes, particularly focused on Complex III, which I will show in a minute in these videos. So this analysis of the movement allowed us to generate new mechanistic hypotheses on the functions of Plant Complex III that we can now experimentally test. So Plant Complex III is unique in that it not only has respiratory functions, but it also has non-respiratory functions. And the question— there has been a question as to whether these two different functions are coupled, functionally coupled, functionally related. So what we saw with our conformational analysis was that complex III has these complex-wide dimer-coordinated movements. And so we hypothesized that these movements form the physical basis for the coupling of Complex III's respiratory and non-respiratory functions. Similarly, our analysis of the movement of Complex III's iron-sulphur cluster domain shown here led us to hypothesise that the movement of this domain is coordinated across the dimers. And again, this would have functional implications not only in plants but in all other organisms as well. So in conclusion, these first high-resolution structures of the plant respiratory complexes and supercomplexes firstly identified several new plant-specific features, furthering our knowledge of plant respiration. They also allowed us to generate new mechanistic hypotheses which we can now experimentally test. And more broadly, they laid the foundation for the development of more specific, more selective agricultural inhibitors to improve crop productivity. So in the future, I am really excited to carry on studying respiration across the tree of life beyond the handful of organisms in which we currently use to study respiration. I am particularly interested in studying respiration in photosynthetic organisms because I think that the interaction between photosynthesis and respiration will reveal new fundamental insights into both respiration and metabolism as a whole. Additionally, photosynthetic organisms, in particular those in the oceans, hold enormous potential as new sources of food and biomaterials. So in order to do this, my goal as an independent investigator will be to examine how the respiratory chain of photosynthetic organisms works at the molecular scale and across photosynthetic clades. So not just in land plants and green algae, but really to survey the variety of biodiverse photosynthetic organisms. So to do this, I will use my expertise in plant bioenergetics and membrane protein biochemistry and structural biology from my postdoc, as well as my expertise in cell molecular and chemical biology from my PhD, to not only further our knowledge of the mechanism, the structure and function of respiratory complexes, but also to lay the foundation for the development of new respiration-based strategies that can improve crop productivity and biomaterial engineering. So with that, I would like to thank everyone in the Letts Lab who has been involved in the project, particularly our group of undergraduate students who really drove the mitochondrial preps in plants, as well as our colleagues on campus, our funding sources. And thank you very much to the committee and the organisers and the mentors. And thank you for your attention.
