Jonikas lab investigates gene for pyrenoid shape and number

Research
Posted on September 6, 2019

Plants, algae and other photosynthetic organisms remove carbon dioxide from the air and incorporate it into starches, in a process known as carbon fixation.  In green algae, which contribute up to a third of global carbon fixation, the efficiency of this activity is greatly enhanced by the presence of an organelle called the pyrenoid.  A new paper by Princeton researcher Martin Jonikas and colleagues, which appeared online in the journal PNAS on August 27, 2019, investigates a gene important for regulating pyrenoid shape and number, and enhances our understanding of this essential component of the global carbon cycle.

In algae, as in plants, the task of carbon fixation is carried out by an enzyme known as Rubisco within the chloroplast. In plants, Rubisco is found throughout the chloroplast, but in algae, Rubisco molecules are clustered together within the chloroplast and form a distinct structure, the pyrenoid.

“Many cellular structures are assembled via phase separation, forming liquid-like droplets in a manner analogous to oil in water,” explain Itakura et al.  The pyrenoid is one of these, made up mainly of phase-separated Rubisco which, together with smaller amounts of other proteins, is surrounded by a starch-based sheath.  In most species, the pyrenoid is penetrated by extensions of the thylakoid membrane, called tubules.  Thylakoid tubules convey concentrated CO2 to Rubisco, greatly improving the enzyme’s efficiency—something that is very important for algae, which live in aquatic environments where CO2 can be hard to access.

Prior studies by Jonikas’ group have shown that the pyrenoid is not a permanent feature of the cell, but instead dissolves during cell division then re-forms as small clusters of phase-separated protein coalesce into a larger mass. Algal chloroplasts normally have only one pyrenoid because, like oil poured onto water, phase-separated protein clusters aggregate together to minimize their exposed surface area.

While conducting a screen for genes affecting pyrenoid function in the green alga Chlamydomonas reinhardtii, Alan Itakura and Leif Pallesen in the Jonikas laboratory uncovered a gene called SAGA1 (Starch Granules Abnormal-1) whose loss causes cells to grow poorly.  When the researchers, including the study’s co-first author, Cindy Chan from Howard Griffiths’ group at Cambridge University, examined the mutant cells, they noticed that saga1 mutants possess multiple pyrenoids—up to 10 per cell.  This was surprising since cells almost always contain just one pyrenoid.  Intrigued, Itakura et al. decided to investigate further.

Because the SAGA1 protein is predicted to contain a starch-binding domain, the researchers first explored whether loss of SAGA1 affects the architecture of the starch plates that make up the pyrenoid sheath.  Indeed, examination of SAGA1-deficient cells under the electron microscope revealed that mutant cells’ pyrenoids have fewer, and abnormally elongated, starch plates in their sheaths.  The authors also found evidence that SAGA1 binds to Rubisco.  Together, these data suggest that SAGA1 helps direct the proper formation of the pyrenoid’s starch sheath, and the attachment of Rubisco to it.  But why would loss of SAGA1 affect pyrenoid number?

“Our results suggest a model where imposing an increased surface area on the liquid-like pyrenoid matrix favors the formation of multiple pyrenoids,” say the authors.  According to this model, phase-separated pyrenoid matrix adheres to the starch plates during pyrenoid formation.  Normally, the starch plates are sized appropriately to create a single pyrenoid, but the elongated starch plates in saga1 mutants pinch off portions of the matrix, resulting in extra pyrenoids. 

Although this model explains why more pyrenoids might appear in saga1 mutants (and in other mutants that result in extra pyrenoids), it doesn’t explain why excess pyrenoids hamper cell growth.  The authors hypothesized that loss of SAGA1 might affect the amount of Rubisco in cells, but found that Rubisco levels are unchanged in saga1 mutants.  This suggests the same amount of protein is simply distributed across multiple pyrenoids.  Importantly, however, Itakura et al. noticed that most of these extra pyrenoids lack a thylakoid tubule network.  Pyrenoids without a thylakoid network would be starved of CO2, suggesting the Rubisco they contain is idled and not contributing to growth.

Whereas the chloroplasts of Chlamydomonas reinhardtii normally contain only a single pyrenoid, cells lacking the gene SAGA1 contain several extras.  In the images above, chloroplasts appear purple, with a representative wild-type chloroplast shown on the left, and a saga1 mutant chloroplast on the right.  Pyrenoid structures appear in green. 

Whereas the chloroplasts of Chlamydomonas reinhardtii normally contain only a single pyrenoid, cells lacking the gene SAGA1 contain several extras.  In the images above, chloroplasts appear purple, with a representative wild-type chloroplast shown on the left, and a saga1 mutant chloroplast on the right.  Pyrenoid structures appear in green. Image credit: Alan Itakura

This work provides a useful new model to explain how a peripheral component, the starch sheath, helps cells regulate their number of pyrenoids.  The authors suggest that such a mechanism may also apply to the biogenesis of other phase-separated organelles such as stress granules.  Jonikas’ lab is currently testing their model, and investigating why the extra pyrenoids lack thylakoid tubules.

Funding:  This study was supported by grants from the National Science Foundation (EF-1105167, IOS-135682 and MCB-1146621), the National Institutes of Health (DP2-GM-119137), and the Simons Foundation and HHMI (55108535) to M.J.; the Biotechnology and Biological Sciences Research Council (BB/M0076391 to H.G. and A.M.); a National Institutes of Health Cell and Molecular Biology Training Grant (5T32GM 7276-42) to A.K.I.; and from the Cambridge IBD International Scholarship, Cambridge Philosophical Society, Cambridge Trust, Lundgren Fund, and Houston Putnum Lowry Prize (Fitzwilliam College) to K.X.C.

Citation: Alan Itakura, Kher Xing Chan, Nicky Atkinson, Leif Pallensen, Liuanyong Wang, Gregory Reeves, Weronika Patena, Oliver Caspari, Robyn Roth, Ursula Goodenough, Alistair McCormick, Howard Griffiths, Martin C Jonikas.  A Rubisco-binding protein is required for normal pyrenoid number, tubule content, and starch sheath morphology in Chlamydomonas reinhardtii.  Proceedings of the National Academy of Science, first published online on August 27, 2019.   https://doi.org/10.1073/pnas.1904587116