Bueyuektas, D. | Search bioRxiv

bioRxiv2025

Role of Chloroplast Lipid-Remodelling Protein 23 During Cold Acclimation in Arabidopsis thaliana

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this adaptation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigate the role of Chloroplast Lipid Remodeling Protein 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and exaggerations in galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrate that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses reveal broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation.

plant biology↗

bioRxiv2025

The evolutionary history of plastid outer envelope proteins - a structure-sequence comparison

Plant metabolism heavily relies on chloroplasts, derived from once free-living organisms. However, how two distinct organisms merged into one remains currently only partially understood. Protein-mediated metabolite exchange across the double-membrane chloroplast envelope is essential for plant cell function. Here, we investigate the evolutionary origins of outer envelope proteins (OEPs) involved in these transport processes. The mosaic nature of the nuclear genome and the deep evolutionary distance since plastid acquisition are major challenges. To address them, we combine sequence-based analyses with emerging structure-based tools, which together enable more sensitive evolutionary comparisons than traditional methods alone. To uncover distinct evolutionary trajectories, we focused on five OEP families: four {beta}-barrel proteins involved in metabolite transport and JASSY, the first published OPDA transporter. We found that the {beta}-barrel proteins were recruited in a stepwise manner and structural homologs of some OEPs point to early recruitment via endosymbiotic gene transfer (EGT). Notably, OEP40 shows a recent structural rearrangement, lacking clear structural homologs in plants other than Arabidopsis, yet retaining sequence conservation across all major land-plant lineages. The JASSY-like family is found across plastid-bearing species, while true JASSY orthologs emerged in embryophytes likely via a stable recruitment of the characteristic lipid-binding domain. Overall, our findings highlight the dynamic nature of the chloroplast outer envelope and show how new functions evolved through structural reshaping and novel domain recruitment. Structure-based approaches thus powerfully complement sequence data, offering more in-depth insight into the evolution of plastid transport systems.

evolutionary biology↗