Conserved and lineage-specific roles of KEA-mediated ion homeostasis in Chlamydomonas

In Arabidopsis thaliana, seamless plastid gene expression and development depend on finely balanced ion homeostasis across the inner envelope (IE) membrane, maintained by the K+/H+ antiporters AtKEA1/2. To assess whether these functions are retained across mono- and polyplastidic representatives of the green lineage, we studied CrKEA1, the sole IE KEA homolog in the unicellular alga Chlamydomonas reinhardtii. Using CRISPR/Cas9, we generated a Cr-kea1 knockout mutant that exhibits impaired photoautotrophic growth, chloroplast deformation, and photoinhibition. Transcriptomics revealed strong induction of ribosome biogenesis genes and reduced abundance of transcripts associated with cell and plastid division. Further RNA analyses confirmed defects in stromal rRNA maturation of Cr-kea1, paralleling observations from Arabidopsis kea1kea2 mutants. Expression of CrKEA1 in Arabidopsis rescued growth and rRNA maturation in At-kea1kea2, demonstrating functional continuity after the ancient divergence between the 2 lineages. Cross-species transcriptomic comparisons further revealed that IE KEA loss elicits both shared and species-specific transcriptional responses: PhANG repression was conserved between algae and plants, whereas activation of the chloroplast unfolded protein response (cpUPR) and reduced expression of genes tied to cell-cycle and plastid fission occurred only in Chlamydomonas. Single-cell time-lapse imaging confirmed that Cr-kea1 exhibits an increased frequency of aberrant cytokinesis, unequal division, and division failure. Our findings demonstrate that while IE KEA transporters fulfill conserved roles in maintaining the conditions for plastid gene expression, their integration into broader cellular networks has diverged between unicellular chlorophytes and embryophytes (land plants). This underscores a lineage-dependent tuning of plastid-nucleus communication shaped by organismal complexity and plastid number.