Fluorescent proteins that Faithfully Reflect the Native Architecture of Chloroplast Nucleoids in Chlamydomonas reinhardtii

Chloroplast genomes are organized into DNA-protein complexes known as chloroplast nucleoids, whose morphology and dynamics are central to genome inheritance. The unicellular green alga Chlamydomonas reinhardtii provides an advantageous model for investigating the mechanisms of chloroplast genome inheritance because it has a simple cellular organization with a single, large chloroplast suitable for live-cell imaging of chloroplast nucleoids. Chloroplast nucleoids can be visualized using the bacterial histone-like protein HU fused to fluorescent proteins (FPs); however, HU-FP fusions can induce nucleoid aggregation in a subset of cells, raising concerns about FP-induced artifacts. Here, we systematically evaluated multiple yellow FP variants fused to HU and examined how each variant affected chloroplast nucleoid morphology in Chlamydomonas. Despite the close sequence similarity among the FP variants, individual variants produced markedly distinct nucleoid morphologies, ranging from globular aggregates to filamentous or dispersed structures. Among the Aequorea victoria-derived FP variants tested, HU-mCitrine most faithfully reproduced the punctate nucleoid pattern observed with chemical DNA staining, whereas HU-mStayGold, whose FP is derived from Cytaeis uchidae, did not induce aggregation but increased the apparent number of nucleoid foci. mCitrine was therefore identified as the most suitable FP for HU-based visualization of chloroplast nucleoids.