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Title: Physiological and molecular determinants of the Chlamydomonas reinhardtii pyrenoid
Author: Meyer, Moritz
ISNI:       0000 0004 2707 3111
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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Aquatic photosynthesis accounts for 50% of the global annual net primary production (NPP), despite frequent low availability and limited diffusion of CO2 in the aquatic milieu, and low affinity for CO2 by the primary carboxylating enzyme, Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Many eukaryotic algae, and a single group of land plants, the hornworts, have an inducible carbon concentrating mechanism (CCM), to overcome these limitations. The efficiency of the CCM is improved when RuBisCO is localised to a subcellular compartment, the pyrenoid, which is hypothesised to act as a diffusion barrier for CO2 . Although the pyrenoid is a major player in global carbon balance (we estimate 10-15% of NPP), it is one of the few remaining prominent cellular features without a precise molecular or physiological definition. Under ambient CO2 , at least 90% of the cellular RuBisCO is packed into a dense matrix, together with the chaperone RuBisCO activase. Thylakoid membranes usually traverse the pyrenoid matrix, and the carboxylating substrate is thought to be delivered to the active sites of the enzyme via a carbonic anhydrase located in the lumen of these thylakoids. The mechanism of aggregation of constituents within the pyrenoid, however, still remains largely unknown. Comprehensive mutant screens have yet to reveal mutants incapable of forming pyrenoids other than those mutants with a defective RuBisCO holoenzyme, whereas DNA microarray studies uncovered little with reference to pyrenoid ultrastructure or aggregation. Taken together, this evidence raises the possibility that the basis of pyrenoid ultrastructure and aggregation lies entirely in sequence variations of RuBisCO itself. This work explored, firstly, the advantages conferred by an active CCM in hornworts and in unicellular algae, compared with the passive CO2 acquisition in most terrestrial plants. A physiological framework to CCM and pyrenoid-based photosynthesis, and isotopic discrimination, was provided by comparing the photosynthetic characteristics of selected bryophytes and algae, differing in chloroplast morphology and degrees of internalisation of gas exchanges. The results showed that on-line, carbon isotope discrimination values were a good indicator of CCM occurrence, as well as liquid-phase diffusion limitation, and biochemical limitations resulting from declining RuBisCO activity and electron transport. The methodology was used to diagnose the presence of an active CCM, and the extent of CO2 leakage. Secondly, the effect of RuBisCO sequence variations on the pyrenoid, and associated CCM, was studied using the model alga Chlamydomonas reinhardtii. The starting premise was the report by Nozaki et al. (2002) that, in some species of the family Chlamydomonaceae, a few amino acid residues within the RuBisCO large subunit (LSU) correlated strongly with pyrenoid formation. The specific roles of seven LSU residues were studied by site-directed mutagenesis. Whilst the mutations reduced the affinity of RuBisCO for CO2 and increased CO2 leakage, compared to wild-type Chlamydomonas, there was no effect on the pyrenoid phenotype. Informed by observations that Chlamydomonas mutants with a hybrid RuBisCO, composed of a native LSU, and higher plant small subunit (SSU), lacked a pyrenoid (Genkov et al., 2010), and that defined SSU alterations were neutral with respect to the pyrenoid (Genkov and Spreitzer, 2006), hitherto unexplored SSU domains were modified. A pyrenoid was successfully restored by replacing jointly the two solvent-exposed α-helices, whereas single α-helix replacements had no effect. However, leakage values indicated that the associated CCM was not fully operative, suggesting important correlates between the RuBisCO SSU and the CCM, besides the conditioning of pyrenoid formation. If the pyrenoid is partly defined by simple sequence variations in the RuBisCO SSU, as suggested by the evidence outlined in this thesis, there is the tantalising possibility that transformation of a biophysical CCM into crop plants could be a tractable approach for the future.
Supervisor: Griffiths, Howard Sponsor: ; Dr. Christian Ries, curator at the National Museum of Natural History, Luxembourg, and president of the Luxembourg Naturalist Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Pyrenoid ; Rubisco ; Carbon concentrating mechanism ; Chlamydomonas ; RbcS