Normal atmospheric CO2 concentration limits photoautotrophic growth rate of most plants and photosynthetic microorganisms, in part because of the inefficiency of the enzyme Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), which has both an oxygenase and a carboxylase function, as well as a low affinity for CO2 (Long et al., 2004, Duanmu and Spalding 2011). Because of this, many photosynthetic organisms have developed strategies to increase the concentration of CO2 around Rubisco, including the well-known C4 photosynthesis and Crassulacean Acid Metabolism (CAM) pathways in terrestrial vascular plants (Niyogi et al., 2015). Some C3-type aquatic photosynthetic microorganisms, including cyanobacteria and microalgae, which face an additional challenge of 10,000-fold slower diffusion rate for dissolved CO2 relative to diffusion in air, have developed a very efficient but very different CO2 concentrating mechanism (CCM) to increase the CO2 concentration around Rubisco.
CCMs that evolved in microalgae and cyanobacteria typically are induced when the supply of dissolved inorganic carbon (Ci) for photosynthesis becomes limiting, thus limiting their metabolic cost when the CCMs are not needed. The essential components of these CCMs include active Ci uptake systems that transport outside Ci for intracellular HCO3− accumulation, at least one internal carbonic anhydrase (CA) that catalyzes rapid interconversion of CO2 and HCO3−, and specific internal compartments to provide a localized elevated CO2 concentration for Rubisco (Moroney and Ynalvez 2007, Spalding 2008, Wang et al., 2011, Freeman Rosenzweig et al., 2017, Mackinder et al., 2017, Mackinder 2018). Much of the knowledge regarding the microalgal CCM has been gained from the well-investigated, model microalga Chlamydomonas reinhardtii (hereafter, Chlamydomonas) (Moroney and Ynalvez 2007, Spalding 2008). When the availability of Ci (inorganic carbon, CO2, and HCO3−) becomes limiting in Chlamydomonas, the induced CCM acts via active Ci uptake systems across both the plasma membrane and the chloroplast envelope to increase internal Ci accumulation, and CAs function to dehydrate accumulated HCO3− and provide elevated internal CO2 concentrations to the pyrenoid, a Rubisco-containing micro-compartment in the chloroplast stroma (Moroney and Ynalvez 2007, Spalding 2008, Wang et al., 2011, Freeman Rosenzweig et al., 2017, Mackinder 2018).
Even though the Chlamydomonas CCM has been extensively studied for many years, we still know little about the limiting CO2 acclimation process. Most candidate Ci transporter genes and CA genes are under control of CIA5/CCM1, a transcription regulator coordinating expression of almost all low-CO2 induced genes (Moroney et al., 1989, Fukuzawa et al., 2001, Xiang et al., 2001, Miura et al., 2004, Fang et al., 2012, Yun et al., 2021, Asadian et al., 2022). Because of the extensive connection of CIA5 to the regulation of most CCM-related gene expression, including regulation of all candidate Ci transporters, CIA5 is often called the “master regulator” of the CCM (Fang et al., 2012).
Much of what we know of CIA5/CCM1 function traces to studies of the characteristics of cia5, a key regulatory mutant defective in CIA5, which was first identified as a High-CO2-Requiring mutant (Moroney et al., 1989, Asadian et al., 2022). The cia5 mutant can grow under high (5% v/v in air) CO2 and grows more slowly than the wild type in low (0.03–0.04% v/v) CO2, but it is unable to grow in very low (0.01% v/v) CO2 and appears to completely lack induction of the CCM (Spalding 2009).
The mutated gene, CIA5, also named CCM1, yields two mRNAs CCM1-A and CCM1-B from a single gene by an alternative splicing between the 3rd and 4th exons (only 2aa difference, 183rd GR substituted to E. CCM1-A: 699aa; CCM1-B also named CIA5: 698 aa) (Fukuzawa et al., 2001, Xiang et al., 2001). CIA5/CCM1 clearly functions as a transcription regulator, and it has been proposed to act specifically as a transcription factor; it has two non-typical Zn-finger (Zn-finger) motifs, which may represent a DNA-binding motif typical of transcription factors. It was confirmed that 2 mol zinc can bind to the N-terminal Zn-finger domain, and that if this protein is mutated with specific replacements, H54Y, C77V and C80V, the zinc binding ability is lost (Kohinata et al., 2008). CIA5 also contains a Gln-rich repeat which might participate in protein-protein interaction and regulation of other eukaryotic transcription factors, and a Gly-rich region with unknown function.
Aside from being a critical upstream transcription regulator of the CCM and other low CO2 acclimation responses and likely requiring posttranslational activation in low CO2, the details of CIA5 function remain undiscovered. We know very little about the genes CIA5/CCM1 directly regulates downstream (Fang et al., 2012), let alone anything about sequences that might be recognized by its putative DNA binding domain. A transcriptome comparison by Fang et al. (Fang et al., 2012) identified a massive impact of CIA5/CCM1 and CO2 on the transcriptome and revealed an array of gene clusters with distinctive expression patterns that provide insight into the regulatory interaction between CIA5 and CO2. Multiple individual gene clusters responded primarily to CIA5, to CO2, or to an interaction between the two (Fang et al., 2012).
Chen et. al (2017) reported the first demonstration that a conserved, C-terminal 130aa acidic domain from CIA5 can function as an activation domain both in Chlamydomonas and in yeast. This conserved acidic activation domain also appears to be indispensable for CIA5 function, as judged from results of complementation experiments with the cia5 mutant (Chen et al., 2017). Together, these observations provided strong suggestive evidence that CIA5 functions, at least in part, through gene-direct activation of gene expression. Whether CO2-responsive genes are specifically targeted for activation by CIA5 alone or by collaboration of CIA5 with specific transcription factors remains a prime question to be answered in future studies (Chen et al., 2017).
Generally, zinc-binding domains including Zn-finger motifs have been shown to function as either sequence-specific nucleic acid recognition motifs or recognition motifs for protein-protein interactions (Kohinata et al., 2008). It was assumed that the CIA5/CCM1 protein could be a DNA-binding protein, since CIA5/CCM1 impacts expression of so many genes and is located in the nucleus (Kohinata et al., 2008). However, when only the zinc-binding domain (N-terminal region) was investigated in southwestern blot analyses using an anti-CCM1 antibody, no signals were detected for DNA-protein complexes, suggesting that CIA5/CCM1 may bind to some unidentified proteins rather than to DNA in the nucleus (Kohinata et al., 2008). The research described here investigated the DNA binding ability of CIA5/CCM1 in vitro.