Biology subjects

Wientjes, E.

Publications and source records attributed to Wientjes, E..

4 recordsLinked to original sources

bioRxiv2025

Charge reversal at the Lhcb2 N-terminus impairs phosphorylation and PSI-LHCII complex formation

State transitions balance excitation-energy distribution between Photosystem I and Photosystem II in higher plants. Stn7-mediated phosphorylation of the N-terminus of the light-harvesting complex II protein Lhcb2 plays a central role in photosynthetic state transitions. However, it remains unclear how the intrinsic charge of this region, independent of its phosphorylation status, influences state transitions and thylakoid membrane organization. Here, we introduced specific charge-altering mutations in the Lhcb2 N-terminus of Arabidopsis thaliana in the lhcb2 knock-out background and analyzed their effects on LHCII phosphorylation, state transition dynamics, PSI-LHCII complex formation, and thylakoid ultrastructure. Substitution of a conserved positively charged arginine with a negatively charged glutamate (R2E) markedly reduced Lhcb1 and Lhcb2 phosphorylation and state transition efficiency, and abolished PSI-LHCII complex formation. In contrast, introducing a negative charge at a downstream position (Q9E) had no detectable effects. Electron microscopy revealed no significant changes in thylakoid organization in either mutant compared to WT Lhcb2 plants. Despite strongly reduced Lhcb1 and Lhcb2 phosphorylation in the R2E mutant, residual state transitions persisted, potentially mediated by Stn7-dependent phosphorylation of other target proteins. Together, these results provide insight into the role of N-terminal LHCII electrostatics in state transitions and thylakoid membrane organization in plants.

plant biology↗

bioRxiv2025

Uniform distribution of photosystems in dark-adapted Synechocystis cells

Photosynthesis in cyanobacteria relies on light capture by photosystem I (PSI), photosystem II (PSII) and the phycobilisome (PBS). While these complexes are thought to be intermixed within the thylakoid membrane, there is also evidence for PSI-enriched and PSII-PBS-enriched microdomains, and their spatial organization is still debated. This organization may further depend on environmental conditions. To study it, a range of methods are available. Cryo-electron tomography offers the highest resolution but is limited in throughput, while super-resolution fluorescence techniques such as Airyscan and structured illumination microscopy provide improved resolution but cannot resolve individual thylakoid membranes. To expand this toolbox, we applied cryo-Expansion Microscopy (cryo-ExM) to dark-adapted Synechocystis sp. PCC 6803 cells. Cells were cryofixed, rehydrated at room temperature and physically expanded in a swellable hydrogel. By expanding cells 5.5-fold, we resolved individual thylakoid membranes in intact cells using confocal microscopy. Immunostaining further allowed simultaneous localization of PSI, PSII and PBS within the expanded thylakoid network. Quantitative analysis of fluorescence covariance revealed a high degree of colocalization among PSI, PSII and PBS, providing no evidence for microdomains under dark-adapted conditions. PBS was excluded only from the neck region between dividing cells, while PSI, PSII and PBS were otherwise distributed throughout the thylakoid membrane. Together, these results establish cryo-ExM as a powerful method for visualizing individual cyanobacterial thylakoid membranes and mapping the distribution of key photosynthetic complexes, thereby complementing existing approaches for dissecting the spatial organization of photosynthesis.

biophysics↗

bioRxiv2023

Expansion microscopy resolves the 3D thylakoid structure

The light-harvesting reactions of photosynthesis take place on the thylakoid membrane inside chloroplasts. The thylakoid membrane is folded into appressed membranes, the grana, and non-appressed membranes that interconnect the grana, the stroma lamellae. This folding is essential for the correct functioning of photosynthesis. Electron microscopy and atomic force microscopy are commonly used to study the thylakoid membrane, but these techniques have limitations in visualizing a complete chloroplast and its organization. To overcome this limitation, we applied expansion microscopy (ExM) on isolated chloroplasts. ExM is a technique that involves physically expanding a sample in a swellable hydrogel to enhance the spatial resolution of fluorescence microscopy. Using all-protein staining, we have visualized the 3D structure of spinach thylakoids with a high level of detail. We were able to resolve stroma lamellae that were 60 nm apart and observe their helical wrapping around the grana. Furthermore, we accurately measured the dimensions of grana from top-views of chloroplasts, which allow for precise determination of the grana diameter. Ultimately, we constructed a 3D model of a complete chloroplast, which provides a foundation for structure-based modeling of photosynthetic adaptations. Our results demonstrate that ExM is a fast and reliable technique for studying thylakoid organization with a high level of detail.

plant biology↗

bioRxiv2021

Photosynthetic light harvesting and thylakoid organization in a CRISPR/Cas9 Arabidopsis thaliana LHCB1 knockout mutant.

Light absorbed by chlorophylls of photosystem II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is the trimeric LHCII. In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of the trimeric LHCII. In absence of LHCB1 the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss leading to a pale green phenotype and growth delay. Photosystem II absorption cross-section was smaller while photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by a lower photosystem I over photosystem II reaction center ratio and by the dephosphorylation of LHCII and photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knock out lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

plant biology↗