Israelievitch, E. | Search bioRxiv

bioRxiv2025

Interplay between high-energy quenching and state transitions in Chlamydomonas reinhardtii: a single-cell approach

Studying cell-to-cell heterogeneity is essential to understand how unicellular organisms respond to stresses. We introduce a single-cell analysis framework that enables the study of intercellular heterogeneity of photosynthetic traits, particularly their interactions within individual cells that have identical genotypes, cellular contexts and histories. Our approach combines single-cell imaging of chlorophyll a fluorescence with machine learning and we study light stress responses in Chlamydomonas reinhardtii as a proof-of- concept. This framework allows us to score the extent of high-light responses such as state transitions (qT) and high-energy quenching (qE), to reveal significant cell-to-cell heterogeneity and to reveal a strong correlation between qT and qE, undetectable in bulk measurements. This study highlights the value of single-cell phenotypic analysis for for investigating light stress responses in unicellular organisms. We detail the key aspects that come into play to generalize the method to other complex stress responses involving multiple traits.

biophysics↗

bioRxiv2024

An optimised protocol for the relative quantification of ATP and polyphosphates in the microalga Chlamydomonas reinhardtii

BackgroundPolyphosphates (polyP) and ATP are phosphate-containing metabolites present in prokaryotes and eukaryotes. PolyP has a wide variety of functions including phosphate and cation storage. ATP is a central metabolite in cellular bioenergetics and the phosphate providing substrate of polyP. In the green microalga Chlamydomonas reinhardtii, polyP synthesis is suggested to buffer ATP concentration, and the role of polyP in energetic metabolism requires further investigation. In this aim, relative quantification of both metabolites is needed. Because ATP and polyP half-lives differ greatly, harvesting generates biases in this relative quantification in current methods. For this reason, we present here a joint protocol optimised to compromise between maximal yield and specific constraints of both assays. MethodsThe optimised method quantifies ATP and polyP from the same C. reinhardtii cell extract after neutral phenol-chloroform extraction. Cells are directly pipetted from the culture to the phenol-chloroform- EDTA extraction mix. After a second chloroform extraction, ATP is quantified directly from the extract, while polyP measurement requires purification by ethanol precipitation. We used one-way analysis of variance or Kruskall-Wallis testing and appropriate post hoc testing to evaluate statistical effects in our results. ResultsWe show that he polyP/ATP ratio of the reference strain CC-4533 in exponential mixotrophic growth is around 65. While the optimised protocol performs as well as specific protocols for either ATP or polyP, the dispersion of the polyP/ATP ratio is twice better than for the separate metabolites. Direct sampling from the culture works better than centrifugation and filtration to maintain physiological conditions and high polyP yield. Using spiking with ATP and polyP, we show that ATP and longer chain polyP are fully recovered but not very short chain polyP. Finally, we show that the polyP chain length distribution extracted from CC-4533 is very broad, reaching up to several thousand P with a mean around 200 P. DiscussionOur protocol improves the precision of relative quantification of ATP and polyP by using neutral phenol-chloroform extraction and allows polyP/ATP ratio calculation from low-density samples and without normalisation. It can be applied to other microorganisms or cells, in a variety of physiological and stress conditions.

biochemistry↗