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ABSTRACT Pyrenoid-based CO₂-concentrating mechanisms (pCCMs) turbocharge photosynthesis by saturating CO₂around Rubisco. Hornworts are the only land plants with a pCCM. Owing to their closer relationship to crops, hornworts could offer greater translational potential compared to the green alga Chlamydomonas, the traditional model for studying pCCM. Here we report the first thorough investigation of a hornwort pCCM using the emerging modelAnthoceros agrestis. The pyrenoids inA. agrestisexhibit liquid-like properties similar to Chlamydomonas, but differ by lacking starch sheaths and being enclosed by multiple thylakoids. We found that the core pCCM components in Chlamydomonas, including BST, LCIB, and CAH3, are conserved inA. agrestisand likely have similar functions based on their subcellular localizations. Therefore, the underlying chassis for concentrating CO₂might be shared between hornworts and Chlamydomonas, and ancestral to land plants. Our study presents the first spatial model for pCCM in a land plant, paving the way for future biochemical and genetic investigations. 

Abstract Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48–72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species. 

Summary Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation.Anthoceros agrestishas been proposed as the model species to study hornwort biology.We have developed anAgrobacterium‐mediated method for the stable transformation ofA. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available.High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenicA. agrestislines expressing the β‐glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed.The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon‐concentrating mechanism, RNA editing and land plant evolution in general. 

Summary Extant land plants consist of two deeply divergent groups, tracheophytes and bryophytes, which shared a common ancestor some 500 million years ago. While information about vascular plants and the two of the three lineages of bryophytes, the mosses and liverworts, is steadily accumulating, the biology of hornworts remains poorly explored. Yet, as the sister group to liverworts and mosses, hornworts are critical in understanding the evolution of key land plant traits. Until recently, there was no hornwort model species amenable to systematic experimental investigation, which hampered detailed insight into the molecular biology and genetics of this unique group of land plants. The emerging hornwort model species,Anthoceros agrestis, is instrumental in our efforts to better understand not only hornwort biology but also fundamental questions of land plant evolution. To this end, here we provide an overview of hornwort biology and current research on the model plantA. agrestisto highlight its potential in answering key questions of land plant biology and evolution.