Structure and function of an inorganic carbon transporter that captures CO2 in green algae.

Abstract

You may have heard the hype about planting one trillion trees to absorb carbon from the atmosphere. However, when it comes to carbon sequestering, green algae are where it’s at. Algae are one of the most efficient organisms in terms of CO2 consumption and do this while growing in an environment where CO2 is a limiting factor. A pair of companion papers (Kono and Spalding, 2020; Kono et al., 2020) in the latest issue of The Plant Journal brings new clues about how they accomplish this feat. Together, algae and cyanobacteria contribute at least half of the world's primary oxygen production. Owing to their unmatched ability to transform CO2 into biomass, algae have recently caught the attention of researchers and entrepreneurs, who are devising strategies to use algae for carbon biosequestration for climate change mitigation and to produce plant-based foods, such as omega-3 and protein sustainably. Algae live in aqueous environments, where CO2 diffuses 10 000 times slower than in air and is slow to equilibrate. This means that water is often easily depleted of CO2 and takes time to gain CO2 from the air. When dissolved in water, CO2 equilibrates with bicarbonate (HCO3−) in a pH-dependent manner. In sea water, the pH is such that dissolved inorganic carbon (Ci) is mainly found in the form of HCO3−. The result of this is a low concentration of free CO2, which is barely sufficient for algal Rubisco to run at a quarter of its maximum velocity. To cope with this limitation, many microalgae and cyanobacteria have developed a CO2-concentrating mechanism (CCM) that efficiently accumulates Ci internally to provide a higher CO2 concentration at the site of Rubisco (Hennacy and Jonikas, 2020). In Chlamydomonas, CCM comprises active Ci uptake (in both CO2 and HCO3− forms) across the plasma membrane and chloroplast envelope. The two companion papers (Kono and Spalding, 2020; Kono et al., 2020) describe the molecular characterization and crystal structure of LC1, a putative plasma membrane Ci transporter involved in the Chlamydomonas CCM. The study comes from the laboratory of Martin Spalding, emeritus professor at Iowa State University (Ames, IA, USA). An expert on the microalgal CCM, Spalding retired in 2016 but still maintains an active research group. The first author of the first paper (Kono and Spalding, 2020) as well as the three joint first authors of the second paper (Kono et al., 2020), Alfredo Kono, Abhijith Radhakrishnan and Tsung-Han Chou, were all graduate students when this work was performed. Kono, who is from Indonesia, joined the Spalding lab in the fall of 2011 with a Fulbright Scholarship and the intention to obtain a Master's degree in Genetics. After his first year, Kono was so intrigued with studying CCM in Chlamydomonas that he requested to switch to a PhD program. Kono graduated with a PhD in Genetics in 2019. The Spalding lab has been working on microalgal CCM for decades, using genetic and physiological approaches to identify and characterize its essential components. Over the last decade, putative Ci transporters and active uptake mediators have been identified, and the group has been trying to understand their molecular function. LCI1 was identified as a candidate for Ci transport by its induction alongside the induction of transport, and by the physiological characterization of a regulatory mutant that failed to induce LCI1 (Ohnishi et al., 2010). Identification of an lci1 mutant in a large-scale screen in another laboratory (Li et al., 2019) offered the opportunity to study this transporter and crystallize the protein for structural analysis in collaboration with Edward Yu, formerly a professor of Chemistry, Physics and Astronomy at Iowa State University. Yu, presently at the School of Medicine at Case Western Reserve University (Cleveland, OH, USA), is a structural biologist mainly focusing on the structural determinations of membrane proteins that are very difficult to crystallize, particularly membrane transporters. Yu and Spalding, who are colleagues and friends, had shared the wish to crystallize one or more of the putative membrane transporters involved in the microalgal CCM. The Yu lab tried initial screenings for crystallization of a few Chlamydomonas transporters, and LCI1 proved to be the most amenable. In the first paper, Kono et al. (2020) performed genetic, growth, and comprehensive Ci-dependent O2 evolution studies of an LCI1 loss-of-function mutant alongside other key CCM mutants at different pHs. Often there is redundancy in the function of microalgal and cyanobacterial Ci transporters, such that the loss of one transporter via mutagenesis frequently results in little phenotypic effect. According to Spalding, this adds to the challenge of studying CO2 or HCO3– transport in small, living cells, which is particularly challenging during rapid, CO2-consuming photosynthesis. The authors developed a subtractive analysis to isolate the impact of a single CCM mutation in the background of a second CCM mutation, which allowed them to observe the functional impact of the loss of LCI1 in the mutant. Their results indicate that LCI1 is involved in active CO2 uptake in a low CO2 range, while it plays a minimal role in a very low CO2 range. The second paper describes the crystal structure of the full-length LCI1 membrane protein, its structural characteristics, and in vivo physiological studies in the lci1 mutant showing the Ci species preference for LCI1. The study represents the first structure determined for any transporter involved in the microalgal CCM. As such, it provides an opportunity for other researchers to develop and test predictions by modifying LCI1 and reinserting the modified LCI1 into the mutant background to test its function. The structure will also allow scientists to develop and test predictions regarding potential complexes and energization pathways regulating the CCM. Based on the results of their two studies, as well as existing knowledge of the Chlamydomonas CCM, the authors developed a hypothetical model to describe the proposed role of LCI1, in coordination with two well-studied Ci uptake systems, LCIA-mediated HCO3− uptake and LCIB-based CO2 uptake (see Figure 1). HCO3– uptake is mediated by the cooperative work between HLA3 in the plasma membrane and LCIA in the chloroplast envelope. On the other hand, the path of LCI1-mediated CO2 uptake from the cytosol into the chloroplast is largely unknown. Further characterization of LCI1-mediated CO2 uptake will support genetic engineering approaches to improve algal photosynthetic growth in bioreactors, for example, for biofuel production. This knowledge will also aid efforts to transfer algal CCM into land plants, such as crops, to boost their photosynthetic capacity.