The pyrenoid, now the subject of intense study, including as part of the CAPP project, is a somewhat mysterious area of cell biology and is as yet not well characterised. Below is what is fairly well-established about the pyrenoid, as well as some existing theories and hypotheses about it, and current areas of research. Understanding the algal pyrenoid is of utmost importance to achieving the aim (and the name!) of the CAPP project.
Pyrenoids are cellular microcompartments found within the chloroplasts of green algal cells (single-celled green algae have just the one chloroplast, thus just one pyrenoid), and are thought to be major components of the algal carbon concentrating mechanism (CCM). They are not membrane-bound, although they do seem to have a defined physical form and are, under certain conditions, surrounded by starch plates. Our current understanding of the pyrenoid is that it is a region of the chloroplast in which most of the RuBisCO in the chloroplast is located, and that it is here that CO2 is concentrated around RuBisCO, allowing the enzyme to operate at a higher efficiency than when dispersed throughout the chloroplast. It is not currently known what other constituents of the pyrenoid there may be besides RuBisCO (see Current Areas of Research below), but some electron micrographs seems to show enlarged tubule-like extensions of the thylakoids into the pyrenoid. It is hypothesised that these may be the route that CO2 takes into the pyrenoid.
It is important to mention the other components that make up the algal CCM. These include a series of putative bicarbonate (HCO3–) transporters for transport into the cell, into the chloroplast and into the thylakoids, as a route into the pyrenoid (CO2 in water forms carbonic acid which dissociates, forming bicarbonate). Additionally, there are several types of carbonic anyhydrase (CAH) known to be present in the cell periplasm (i.e. the space just outside the plasma membrane but within the cell wall), the cytosol, the stroma and the thylakoid lumen. These catalyse interconversions between CO2 and HCO3– which is an important factor in carbon concentrating: CO2 can diffuse more or less freely across lipid membranes as a small gaseous molecule, so cannot be accumulated within a membrane-bound cell or cellular compartment in this form. CAH-mediated conversion to bicarbonate, therefore, allows carbon to be accumulated to a higher level, and allows its transport to be more regulated. However, RuBisCO requires CO2, not bicarbonate, as its substrate, thus other CAHs are required to convert the bicarbonate back to CO2 for fixation. So the three major components of the algal CCM are: a pyrenoid, a series of transporters and a series of carbonic anhydrases.
Current Areas of Research
Research aiming to elucidate details of the algal (Chlamydomonas) pyrenoid currently taking place in Prof. Howard Griffiths’ lab includes:
- What holds the pyrenoid together? Investigations are currently taking place to establish whether RuBisCO molecules themselves can self-adhere, creating effectively a raft of RuBisCO which appears as the pyrenoid, or whether other proteins are involved in holding together the pyrenoid.
- Localisation studies using GFP-tagging to establish the localisation of the transporters associated with the pyrenoid.
- Ongoing mutagenic studies to establish the necessary and sufficient genetic components for pyrenoid formation. It is already known through the formation of hybrid RuBisCO small subunit mutants (see Experimental Techniques) that such genetic sequences include those for the 2 α-helices on the small subunit of RuBisCO. Further research is now being carried out to establish the minimal amount of genetic change, and the nature of this change, that would be required to allow the formation of a pyrenoid in plant cells.
- Study of knockdown mutants (using miRNAs) of various transporters to establish their importance in the algal CCM.
- Growth of pyrenoid-positive and pyrenoid-negative Chlamydomonas strains under differing light and ambient CO2 conditions in order to establish the inducibility of the CCM components, and the extent of the advantages to the alga (in terms of growth and survival) under low CO2 conditions of having a CCM.