The Impact of Secondary Endosymbiosis on PSII Architecture : Functional Analysis of CP29 in Euglena gracilis

The photosynthetic machinery of Euglena gracilis, a secondary alga with a complex
evolutionary background, exhibits notable deviations from the canonical systems
characterized in green plants and algae. This study explores how E. gracilis adapts its
photosystem II (PSII) architecture and regulatory mechanisms in the absence of CP26
(Lhcb5), a canonical light-harvesting subunit.
Despite extensive characterization of regulatory mechanisms, including NPQ, alternative
electron flows, and state transitions in model species like Arabidopsis and
Chlamydomonas, such processes remain largely overlooked in photosynthetic protists. The
evolutionary mosaicism of E. gracilis likely resulting from endosymbiotic gene transfers
has likely led to unique modifications in its photosynthetic regulation.
This divergence is exemplified in E. gracilis , which lacks the typical CP26 protein of plant
PSII but possesses a divergent family of red-shifted chlorophyll a-binding antenna proteins
(LHCE), which assemble into specialized pentameric structures under low light. To better
understand how these features impact PSII organization and regulation, we employed
cryo-electron microscopy and proteomic approaches.
Our structural analysis shows a stable dimeric PSII‒LHCII supercomplex in wild-type cells,
composed of CP29 and three LHCII trimers per PSII core. Notably, CRISPR-Cas9-mediated
knockout of CP29 led to a complete disruption of this supercomplex in clear-native PAGE
analysis, resulting in PSII cores dissociated from LHCII trimers. Yet, surprisingly, biophysics
analyses revealed that the CP29-KO strains maintained normal growth and photosynthetic
parameters (ΦPSII, PSII antenna size and electron transport rates (rETR)) under a range
of light intensities (white and far-red light).
Closer inspection revealed that CP29-deficient cells exhibited an altered fluorescence
responses following far-red light exposure, suggesting a subtle yet specific role for CP29
in facilitating state transitions and maintaining antenna-PSII connectivity. These findings
hint at compensatory mechanisms within the light-harvesting apparatus.
Among potential candidates, LHCE9-3 emerged as a possible functional substitute for
CP29. Given its spatial proximity to the PSII core in structural data and its classification
within the monomeric LHCE family, we hypothesized that LHCE9-3 may compensate for
the absence of CP29 and CP26.