Supervisor: Prof. Howard Griffiths
Importance of the area of research
Ribulose-1,5-Biphosphate-carboxylase-oxygenase (Rubisco) is arguably the most important enzyme on Earth, feeding and fuelling mankind through carbon assimilation and helping to sequester anthropogenic CO2 emissions. The enzyme, is seemingly inefficient and aquatic photosynthesis face is also constrained by slow diffusion of CO2 in the aqueous phase. Most cyanobacteria and marine microbes have developed CO2-concentrating mechanisms (CCM) to enhance the operating efficiency of Rubisco. An intracellular pool of inorganic carbon (Ci), usually as HCO3-, accumulates within specialised micro-compartments containing most of the cellular Rubisco (carboxysomes in cyanobacteria or pyrenoids in eukarytic algae). Aquatic CCMs and their associated micro-compartments are present in most microbial algal lineages, and have evolved independently many times. We know little about the phylogeny and physiology of these processes in eukaryotes, which annually fix perhaps 20- 30% of global primary productivity. The proposal offers the opportunity to explore the ecology, molecular physiology and phylogeny of pyrenoid formations in these amazingly productive micro-organisms.
The objective of this proposal is to reconstitute the evolutionary history of the pyrenoid (and hence CCM) using the molecular clock, found within Rubisco large and small subunit sequences, in relation to palaeohistorical atmospheric CO2 concentrations over the past 1 billion years. Initially the programme will focus on the distribution of the pyrenoid in green algae, and then extend to include additional photosynthetic microbial lineages. The ‘green’ lineage diverged maybe 1 BYA into Chlorophyta and charophytes, the sister group to vascular plants, and there have been multiple occurrences of pyrenoid-containing clades.
What the student will be doing
Firstly, a phylogeny of nuclear and plastid encoded Rubisco genes (small and large subunits, respectively) will be conducted on 100+ green algal sequences to infer the history of gains and losses of the pyrenoid in this clade. Missing sequences will be recovered from culture collection material. Presence/absence of a pyrenoid will be mapped onto the phylogeny, and the co-evolution of the two genes will also be analysed. The study will also extend to other proteins known to be uniquely associated with the pyrenoid, to investigate evolutionary divergence
Secondly, small and large subunit sequence alignments will be tested for positive selection at the level of each residue. Initial emphasis will be on residues belonging to the two Rubisco small subunit alpha helices, which have been shown previously to be implicated in pyrenoid formation.
Training that will be provided
Growth and maintenance of algal/marine microbial cultures . Training will include cDNA library generation, molecular sequence analysis and phylogeny, informed by literature meta-analysis and computational analysis. Methods will include Maximum Likelihood and Markov Modelling to infer the probability of absence/presence for each branch of the phylogeny. Physiological characterisation of CCM induction physiology and pyrenoid ultrastructure (electron microscopy).
A background in plant and or microbial physiology, or molecular ecology.
- Meyer, M.T., Genkov, T., Skepper, J.N., Jouhet, J., Mitchelle, M.C., Spreitzer, R.J. & Griffiths, H. 2012. Rubisco small-subunit alpha-helices control pyrenoid formation in Chlamydomonas. Proc Natl Acad Sci USA, 109, 19474-19479, doi: 10.1073/pnas.1210993109.
- Meyer, M.T. & Griffiths, H. Origins and diversity of eukaryotic CO2-concentrating mechanisms: lessons for the future. J Exp Bot 64, 769-786, doi: 10.1093/jexb/ers390.
- Villarreal, J.C. & Renner, S.S. Hornwort pyrenoids, carbon-concentrating structures, evolved and were lost at least five times during the last 100 million years. Proc Natl Acad Sci, 109, 18873-18878, doi: 10.1073/pnas.1213498109.