IVAN HURTADO CACERES¹, Denilson Fernandes Peralta², Cláudia Maria Furlan¹

Hornworts (Anthocerotophyta) are the least diverse and most understudied lineage of bryophytes, yet they retain ancestral traits such as pyrenoid-bearing chloroplasts. These structures, well known in algae, support carbon-concentrating mechanisms (CCMS) that enhance photosynthetic efficiency under CO2-limited conditions. While pyrenoids have been functionally characterized in algal models, their contribution to land plant metabolism—especially across the haploid and diploid generations—remains poorly understood. Here, we investigate metabolic specialization between the haploid gametophyte and diploid sporophyte generations in Anthoceros hispidus, focusing on the potential role of pyrenoid-associated photosynthesis in shaping carbon allocation and metabolic compartmentalization. An untargeted gas chromatography–mass spectrometry (GC-MS) approach was applied. Specimens were collected from the Instituto de Pesquisas Ambientais (IPA, São Paulo), freeze-dried, and extracted using a chloroform:methanol:water protocol. Polar phases were derivatized with MSTFA and analyzed by GC-MS. Feature detection and spectral annotation were conducted via GNPS, with curation against GMD, NIST, and MassBank databases, with complementary validation through authentic standards. Annotation confidence followed the Metabolomics Standards Initiative (MSI) guidelines, achieving level 2 for spectral matches and level 1 for compounds confirmed by retention time and fragmentation spectra. To ensure analytical robustness, features with blank/sample intensity ratios >1:3 were removed. Data were normalized by total ion current (TIC), cube root-transformed, and Pareto-scaled. Multivariate (PCA, OPLS-DA) and univariate (volcano plot) analyses were conducted using MetaboAnalystR. Pathway enrichment analysis was performed through KEGG using Arabidopsis thaliana as the reference. A total of 63 polar metabolites were annotated, 27 of which displayed statistically significant differences between the two generations (VIP ≥ 1; p < 0.05; |log2FC| ≥ 1). Among these, 19 were more abundant in the gametophyte and 8 in the sporophyte. Sugars were the most abundant chemical class across both generations, though not differentially accumulated, suggesting possible translocation of photoassimilates from gametophyte to sporophyte through haustorial cells. The gametophyte exhibited enriched levels of metabolites associated with carbohydrate metabolism and carbon fixation, consistent with the exclusive presence of pyrenoids in its chloroplasts and their proposed role in sustaining high photosynthetic capacity and energy demand. By contrast, the sporophyte showed limited accumulation of polar metabolites, indicating partial physiological autonomy but metabolic dependence on the gametophyte. Altogether, our findings provide novel insights into metabolic differentiation associated with alternation of generations in hornworts. Altogether, our findings offer new insights into metabolic differentiation associated with alternation of generations in hornworts. They also suggest that pyrenoid-based CCMs contribute to generation-specific specialization in carbon metabolism, supporting broader hypotheses on the evolution of resource allocation and photosynthetic strategies in early diverging land plants.

Agradecimentos: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2021/06227).