Super-resolution expansion microscopy reveals nanoscale protein domains and CO 2 -dependent remodeling of Chlamydomonas pyrenoid-traversing membranes

Garde, A.; Wu, H.; Jonikas, M.

doi:10.64898/2026.06.11.731689

Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.06.11.731689

Super-resolution expansion microscopy reveals nanoscale protein domains and CO 2 -dependent remodeling of Chlamydomonas pyrenoid-traversing membranes

Garde, A.·Wu, H.·Jonikas, M.

View original source ↗Export metadata ↓

Abstract

Within the algal carbon-assimilating organelle, the pyrenoid, specialized traversing membranes perform the essential function of delivering concentrated CO2 to Rubisco. In Chlamydomonas reinhardtii, these membranes consist of peripheral cylindrical tubules that connect to a central reticulated region. However, due to resolution limitations, the spatial distribution of their structural and functional proteins has remained unclear. Here, we achieve an [~]11-fold improvement in resolution by combining ultrastructure expansion microscopy with super-resolution instantaneous structured illumination microscopy, revealing protein localizations and condition-dependent remodeling of these membranes. At air levels of CO2, the tubule-initiating protein SAGA1 forms narrow rings at the pyrenoid edge, the tubule-extending protein MITH1 surrounds the peripheral tubules, and the putative transporter BST4 surrounds tubules more centrally, suggesting that the cylindrical tubules contain multiple distinct protein domains. The CO2-delivering carbonic anhydrase CAH3 localizes to the inner face of the central reticulated region, suggesting that this region is specialized for CO2 delivery. CAH3 remains in the reticulated region at high CO2, suggesting that the cell maintains a minimal CO2-delivery apparatus even when dispensable. Finally, at high CO2, cylindrical tubule diameter narrows, and MITH1 relocalizes throughout the pyrenoid-traversing membrane network. Together, our study elucidates sub-pyrenoid protein organization and CO2-dependent reorganization.

Explore related subjects

Plant Biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Garde, A., Wu, H., Jonikas, M.. 2026-06-15. Super-resolution expansion microscopy reveals nanoscale protein domains and CO 2 -dependent remodeling of Chlamydomonas pyrenoid-traversing membranes. https://doi.org/10.64898/2026.06.11.731689

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Enhanced production of nitrogenase components in Nicotiana benthamiana through co-expression with Bacterioferritin A

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI

Plant Biology↗

bioRxiv2026

Seasonal climatic impacts on orchid productivity in an urban ecosystem

Context: The global diversity hotspot in Southwest Australia has >480 orchids facing increasing threats from climate extremes, fire and habitat decline. Aims: To develop effective and consistent tools for measuring climate impacts on productivity in a diverse urban orchid community. Methods: Annual variations in flower and seed production for 17 orchids were determined using thousands of records over a decade with extreme climate variability. Key results: Rainfall deficits and temperatures in autumn, winter and spring increased substantially over 125 years. Seasonal climate anomalies reduced flowering and seed production for orchids, but this varied between species and seasons. These effects were summarised by climate response (CRI) and sensitivity (CSI) indexes. Early or late flowering species were most vulnerable to seasonal drought, but warm dry conditions promoted visually deceptive pollination. CRIs were strongly correlated with orchid pollination syndromes and flowering times. Effects on mycorrhizal fungi and pollinators were also observed. Extrapolating climate trends to 2100 predicted further impacts on orchid productivity (5-40%). Conclusions: Orchid climate responses were substantial, complex and deeply integrated with key traits such as pollination, phenology and fire responses. Implications: Research in an urban climate observatory produced a climate analysis framework potentially relevant to all orchids and other biota.

Plant Biology↗

bioRxiv2026

Biuret inhibits Arabidopsis root growth through an active, reversible, and genetically tractable developmental response

Biuret, a nitrogen-rich by-product of urea and a common contaminant of urea-based fertilisers, has long been considered a passive phytotoxin, affecting plant performances. Yet its effects on root development and the existence of endogenous mechanisms of perception or tolerance remain largely uncharacterised. Here we combined physiological, developmental, genetic and transcriptomic approaches to investigate the response of Arabidopsis thaliana to biuret. Biuret inhibited primary root growth in a dose-dependent manner by reducing meristematic cell division rather than cell elongation, and concomitantly impaired shoot growth by limiting leaf expansion. This root inhibition was reversible upon biuret removal and was accompanied by increased auxin-responsive (DR5) and decreased cytokinin-responsive (TCS) outputs at the root apex, consistent with a regulated remodelling of meristem activity rather than purely cumulative damage. A forward genetic screen identified the biuret-resistant mutant bir29, which sustained root and inflorescence development under inhibitory concentrations. Using {superscript 1}N-labelled biuret, we showed that resistance occurred without any change in biuret influx or accumulation, uncoupling sensitivity from exposure. Whole-genome transcriptomics revealed that bir29 fails to execute the wild-type response, neither repressing the cell-cycle machinery nor deploying the stress-associated programme induced by biuret. Genetic characterisation linked resistance to multiple genomic loci required for full resistance. Together, the results indicate that biuret triggers an active, reversible and genetically tractable developmental response, suggesting that this xenobiotic compound is integrated into endogenous signalling networks. Significance StatementBiuret, a poorly metabolised contaminant of urea fertilisers, is generally regarded as a passive phytotoxin, yet we show that it inhibits Arabidopsis root growth through a reversible and genetically tractable developmental response, accompanied by reorganised auxin and cytokinin signalling, rather than through cumulative chemical injury. The isolation of the resistant mutant bir29 suggests that plants integrate this xenobiotic molecule into endogenous signalling networks, reframing biuret as an informative probe of root developmental regulation.

Plant Biology↗