PSII REGULATION IN EUGLENA GRACILIS: FUNCTIONAL INSIGHTS INTO CP29 AND LHCE9-3

This project seeks to investigate photosynthetic regulation in Euglena gracilis. While significant progress has been made in understanding photosynthesis in green lineage organisms such as flowering plants and green algae, on the other hand the regulatory mechanisms in photosynthetic protists, including euglenophytes like E. gracilis, remain poorly understood. Recent advancements in the study of photosynthesis have uncovered diverse regulation mechanisms in model organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. These include alternative electron transfers, non-photochemical quenching (NPQ), and state transitions (qT) that balance energy distribution between photosystems. While such regulatory mechanisms in secondary algae like E. gracilis are far less explored. The unique evolutionary history of E. gracilis, involving the integration of genomes from both host and symbiont, has likely driven the development of distinctive metabolic pathways and deep reorganization of photosynthetic regulation. This entails in E. gracilis the loss of some canonical subunits such as CP26/Lhcb5 chlorophyll a/b-binding protein of plant photosystem (PS) II, as well as the presence of a unique family of light-harvesting complexes (LHCE), which mainly contains red-shifted chl a which assembles into a pentameric antenna complex in low-light environments. Despite the absence of CP26, Euglena cells assemble a dimeric PSII-LHCII supercomplex comprising CP29/Lhcb5 and three LHCII trimers (LHCII3) per PSII core. Using a CRISPR-Cas9 gene editing approach, CP29 knockout (KO) mutant strains were generated. In these strains, no PSII-LHCII supercomplexes were detected; instead, only PSII cores and free LHCII3 were identified after Clear-Native PAGE analysis of total membrane protein preparations solubilized with alpha-dodecyl-maltoside. These results suggest that the loss of CP29 compromises the stability of the PSII-LHCII supercomplex in vivo. Interestingly, despite this structural destabilization, CP29-KO strains did not exhibit any detectable growth or photosynthetic defects under either light-limited or high-light conditions. However, by monitoring changes in maximum fluorescence emission at room temperature following short-term far-red light exposure (which reflects the interaction dynamics between the antenna complex and PSII), a potential impairment in state transitions was observed. These findings point to a more specific functional role for CP29 in the regulation of energy distribution and in the general stability of PSII-LHCII supercomplexes. Building upon this, our study suggests that E. gracilis has evolved a stable PSII-LHCII3 supercomplex in the absence of CP26, and that its assembly and photoprotective capacity are not fully compromised by the additional loss of CP29. This raises the possibility that other light-harvesting proteins may compensate functionally in the absence of CP29. In this context, we investigate the role of LHCE9-3, which might partially substitute for CP29 in the PSII-LHCII complex, potentially contributing to its stabilization and function. To explore this hypothesis, we are currently examining whether novel monomeric LHCE proteins, such as LHCE9-3, cooperate with or compensate for CP29 in maintaining PSII-LHCII integrity. Moreover, preliminary data point to a potential involvement of the LHCE9-3 subunit in state transitions, based on its genotypic proximity to the pentameric light-harvesting complexes (LHCE) and its observed position within the PSII supercomplex. To test this, we employed double-stranded RNA (dsRNA) interference to specifically downregulate LHCE9-3 expression in E. gracilis. This strategy enables a functional analysis of LHCE9-3 by monitoring changes in maximum fluorescence emission following far-red light exposure, thereby clarifying its role in dynamic PSII regulation.