Ruaud, S.; Noetzold, S. I.; Waller, M.; Galbier, F.; Mousavi, S.; Charran, M.; Zeeman, S.; Baily, A.; Baroux, C.; Hippler, M.; Wicke, S.; Szoevenyi, P.

Biophysical carbon concentrating mechanisms (CCMs) operating at the single-cell level have evolved independently in eukaryotic algae and a single land plant lineage, hornworts. An essential component for an efficient eukaryotic CCM is a pyrenoid whose biology is well-characterized in the unicellular green alga, Chlamydomonas reinhardtii. By contrast, pyrenoids and CCM are little understood in hornworts. Here, we investigate the molecular underpinnings and dynamics of hornwort pyrenoids. We do so by studying the subcellular localization of candidate proteins homologous to essential CCM genes in C. reinhardtii and assessing their mobility kinetics in the hornwort model Anthoceros agrestis. We provide evidence that an EPYC1 analog and the Rubisco co-localize in the pyrenoid but pyrenoids seem less dynamic in A. agrestis than in C. reinhardtii. We further found that a carbon anhydrase homolog (CAH3) localizes to the pyrenoid, while an LCIB-like homolog is less intimately linked to the pyrenoid than in C. reinhardtii. Our results imply that the pyrenoid-based CCM of hornworts is characterized by a mixture of Chlamydomonas-like as well as hornwort-specific features which is in line with its independent evolutionary origin. Using these observations, we provide a first mechanistic model of hornwort CCM.