Molecular underpinnings of hornwort <scp> CO ₂ </scp> concentrating mechanisms: subcellular localization of putative key molecular components in the model hornwort <i>Anthoceros agrestis</i>

Biophysical CO₂ concentrating mechanisms (CCMs) operating at the single-cell level have evolved independently in eukaryotic algae and a single land plant lineage, hornworts. An important 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 Paton. We provide evidence that an essential pyrenoid component 1 analog and the ribulose 1,5-bisphosphate carboxylase/oxygenase colocalize in the pyrenoid, but pyrenoids seem less dynamic in A. agrestis than in C. reinhardtii. We further found that a carbonic anhydrase homolog localizes to the thylakoid membranes between the pyrenoid subunits, while an limiting CO₂-inducible B-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 and hornwort-specific features, which is in line with its independent evolutionary origin. Using these observations, we provide a mechanistic model of hornwort CCM.