Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (volvocales)

The morphology of the pyrenoid and the physiology of the CO2-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969 , Cd. macrostellata Lund SAG 72.81, Cd, bipapillata Bourrelly SAG 11-47, an d Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are clo sely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. au gustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, e lectron-dense pyrenoid matrix surrounded by starch granules was present, an d ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) mo lecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of CI, insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher co ncentration of Rubisco molecules than the thylakoid region (thylakoid and s troma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chla mydomonas strains examined in this study and that of Cd. reinhardtii Dangea rd. In all six strains examined, the presence of the CCM was indicated by r elatively high photosynthetic affinities for CO2 (low values of K-0.5(CO2)) . However, differences in the inorganic carbon (Ci) pools were recognized i n relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cn. rad iata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2-3 similar to the two pyrenoid-less, CCM-containing strains of C hloromonas previously examined (E. Morita et al., 1998, Planta 204. 269-276 ). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydo monas/Chloromonas strains and the pyrenoid-less Chloromonas strains previou sly investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with hi gh bootstrap values. Based on the tree topology resolved, it was inferred t hat loss of the typical pyrenoids accompanied by a decrease in intracellula r Ci pools might have taken place independently in the two groups.