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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.

Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp., Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, and Spaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of the Folioceros, Dendroceros, and Notothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. In Megaceros flagellaris and M. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids. Anthoceros fusiformis and Phaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region in A. fusiformis and P. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20099号 / 生博第359号 / 新制||生||47(附属図書館) / 33215 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 福澤 秀哉, 教授 佐藤 文彦, 教授 河内 孝之 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.