Functional Characterization of the Transcription Factor RR-B in Chlamydomonas: Integrating Omics and Phenotypic Landscapes to Elucidate Regulatory Roles

Microalgae are central to sustainable biotechnological applications, including biofuel production, carbon sequestration, and the generation of high-value metabolites. Their ability to remodel metabolism in response to environmental stress, particularly nutrient deprivation, makes them ideal systems for studying cellular adaptation. This work integrates recent advances on the molecular and regulatory mechanisms governing stress responses, lipid metabolism, and phytohormone signaling in Chlamydomonas reinhardtii. Under nitrogen deprivation and other stressors, microalgae reallocate carbon fluxes toward storage lipids and modulate transcriptional networks controlling cell division, chloroplast maintenance, and biomass accumulation. Through transcriptomic, lipidomic, and genetic analyses of cytokinin-related signaling components—including AHK, AHP, and RR-B homologs—we reveal that C. reinhardtii possesses a proto-cytokinin signaling module that influences lipid homeostasis, stress tolerance, and chloroplast resilience. Characterization of the rrb-1 mutant demonstrates that disruption of the RESPONSE REGULATOR TYPE-B gene causes misregulation of lipid metabolism, altered thylakoid composition, impaired cilia formation, and a constitutive palmelloid phenotype, underscoring its regulatory role in carbon partitioning and stress adaptation. Comparative genomic and metabolomic analyses on independent CRISPR mutants indicate that algal cytokinin-like pathways operate through tRNA-derived cytokinins and lack canonical CHASE domain receptors, suggesting that land plants later co-opted and specialized this ancestral system. Collectively, these findings illuminate the interconnected regulation of nutrient sensing, phytohormone signaling, and metabolic reprogramming in microalgae, providing mechanistic insights that can inform metabolic engineering and the optimization of algal cultivation for industrial and environmental applications.