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Building a CO2-concentrating mechanism


Guest post by Steven Burgess


Researchers from the Universities of Edinburgh, York, Cambridge and Princeton have demonstrated a key step towards reconstructing a CO2-concentrating mechanism (CCM) used by green algae in plants. They were able to form a hybrid carbon fixing complex with parts from algae and plants in vitro as a proof of principle for crop engineering. This work is part of an international collaboration that aims to test predictions that increasing the CO2 concentration in leaves, with a system adapted from algae, will enhance photosynthetic performance, as well as water and nutrient use efficiency.


Alistair McCormick, a lecturer at the University of Edinburgh, and lead PI on the publication, explains: “In algae, CO2 levels can be raised by 50-100 times above ambient air, which enhances the efficiency of CO2 assimilation. Introducing a functional algal  CCM into a C3 higher plant is predicted to lead to significant enhancements in photosynthesis, water use, and nitrogen availability.”


Over the last century food supplies were able to match a growing global population through large increases in yield. Breeders primarily increased yields by improving both the efficiency with which plants capture photosynthetically active radiation over a growing season, and how they partition biomass into grain. These approaches have proved so effective that there is little additional room for improvement. This is a problem as it is predicted a doubling of food supplies will be required by 2050 to meet demand as the global population increases from around 7.5 to 11 billion.


However, evidence suggests the photosynthetic efficiency of crops remains well below limits providing an attractive target for improving yields. Genetic manipulation of plants to increase the amount of carbon fixed resulted in increased biomass in Arabidopsis (here) , tobacco (also here, here and here), tomatoes, corn, and wheat. Although the process may not be entirely straight forward, as similar approaches in rice have failed to yield benefits, this work provides a demonstration of what can be achieved, and by supplying providing more carbon it may be possible for breeders to convert this into yield benefits. Indeed, this approach is not new, as many photosynthetic organisms have evolved mechanisms to perform the same trick.


The metabolic reactions of photosynthesis developed when the earth’s atmosphere had much lower concentrations of oxygen. But as atmospheric oxygen concentrations rose, with the spread of photosynthetic organisms across the globe, it led to competitive inhibition of the key enzyme involved in carbon fixation known as Rubisco. As a result, CCMs repeatedly evolved in algae and plants to suppress the inhibitory effect of Oxygen at a small energetic cost.


Today, CCMs contribute to the success of some of our most productive crops, such as Sugarcane and Corn. Mathematical modelling of photosynthetic metabolism suggests that under warm environments introducing CCMs into crops such as rice and wheat may boost yields. However, rice and wheat, the two most valuable crops in the world do not possess CCMs. Consequently, there are ongoing efforts to engineer crops with CCMs from cyanobacteria, C4 and CAM plants,  “given our current challenges with food security and climate change, all CCM avenues definitely should be explored,” says Alistair. “Plant-based CCMs, such as C4, could produce similar improvements in yields as predicted for algal (or cyanobacterial) CCMs.. One of the presumed advantages of the latter two, is that they occur in single cells, so could be introduced directly into C3 mesophyll cells. In contrast, engineering a functional C4 pathway may require additional modification of C3 leaf cellular architecture to match the two cell Kranz anatomy typical of C4 plants.”

A key part of the algal CCM is a subcellular structure known as a pyrenoid. “Pyrenoids are fascinating, proteinaceous compartments found inside chloroplasts” says Alistair. “They primarily consist of the key CO2 assimilation enzyme Rubisco and have liquid-liquid-like properties, such that they can dynamically disassemble and reassemble when needed (e.g. under conditions of low CO2 availability).” They come in many different shapes and sizes, and have been found in a variety of organisms including  algae, diatoms, dinoflagellates, as well as a group of ancient, non-vascular plants called hornworts. Due to the size of pyrenoids, which can take up a substantial portion of the chloroplast, they were one of the first subcellular compartments to be identified over 200 years ago.

The currently hypothesised composition of the CCM found in Chlamydomonas reinhardtii. 1= Extracellular environment. 2= Plasma membrane. 3= Cytoplasm. 4= Chloroplast membrane. 5= Stroma. 6= Thylakoid membrane. 7= Thylakoid lumen. 8= Pyrenoid. Image by Moritz Meyer, CC BY-SA 3.0

However, engineering a pyrenoid into vascular plants is not a simple task. “Despite their ecological importance” says Alistair, “very little is known about the mechanisms regulating pyrenoid formation and how these might differ between species.”


Therefore McCormick and colleagues have chosen to characterize the CCM from the model alga, or “green yeast” Chlamydomonas reinhardtii. “[It is] the most well characterised algal CCM“ says Alistair. “We want to understand better how several known and recently identified components of the Chlamydomonas CCM work to regulate pyrenoid assembly and function, and use these components to build a minimal, but fully functional pyrenoid-based CCM in a higher plant (Arabidopsis).”


The idea of “minimal systems” is a powerful concept in synthetic biology, most famously in the case of the artificial bacterium created by researchers at the Craig Venter Institute in an effort to define the minimal set of genes required for life. While there is a conceptual model of the algal pyrenoid, an understanding of whether these components are sufficient is lacking. Reconstructing an algal CCM in a heterologous system, such as Arabidopsis , can address this.


“Together with the ground breaking gene discovery and characterisation work done by Martin Jonikas, Luke Mackinder and Howard Griffiths in Chlamydomonas, our efforts to build an algal CCM in Arabidopsis have already helped us to refine our understanding of the location of putative CCM components, how they function, and their compatibility with higher plants” says Alistair. “Ultimately, the best way to understand how an engine works is to try and rebuild it!”


While at least 89 proteins have been identified in the Chlamydomonas pyrenoid including rubisco interacting proteins and inorganic carbon transporters, it is believed a much smaller number are essential for functioning. McCormick and colleagues showed that essential pyrenoid component 1 (EPYC1) was able to interact with Rubisco in vitro and phase-separate into protein aggregates. In addition they showed EPYC1 could be expressed in Arabidopsis and be targeted to the chloroplast. However, EPYC1 expression did not result in rubisco aggregation in planta, most likely because EPYC1 was subject to proteolytic degradation.


Demonstrating EPYC1 is sufficient for rubisco aggregation is an important first step, as rubisco aggregates are necessary to generate a high concentration of CO2. However, moving forward, Alistair highlights a two parts that are missing from a minimal CCM: “ Pyrenoids are traversed by tubules, seemingly specialised thylakoid membranes. We still don’t understand how these enigmatic tubules integrate into pyrenoids or their exact functional role, although we believe that they are involved in supplying CO2 to Rubisco.” says Alistair.  “We also lack sufficient data on the kinetics of inorganic carbon uptake (e.g. the putative CO2/HCO3- transporter components in Chlamydomonas).”


As a result, he plans to continue using the Chlamydomonas mutant library and screening approaches developed by Martin Jonikas and Luke Mackinder to uncover these parts. “In the medium term we hope to build proto-pyrenoid structures in higher plants and work towards having a functional, synthetic algal-based CCM system” says Alisitair “If we succeed… we aim to move our work from Arabidopsis into a useful crop plant.”


Steven Burgess is a postdoctoral researcher at the University of Illinois at Urbana-Champaign. He works for the Realizing Increased Photosynthetic Efficiency (RIPE) project and is coordinating the Plant Synthetic Biology Network on the American Society for Plant Biology website Plantae. You can follow him on twitter @sjb015

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