Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici

Single-cell C₄ (SCC4) photosynthesis in Bienertia sinuspersici requires the formation of dimorphic chloroplasts within a single photosynthetic cell, yet the early developmental transitions preceding this differentiation remain poorly defined. Here, we conducted staged transcriptome profiling of young, intermediate, and mature leaves to characterize transcriptional dynamics associated with early SCC4 establishment, with selected expression patterns validated by qRT-PCR. From 55,257 expressed transcripts, genome-guided filtering identified 199 genes strongly enriched in young leaves and markedly repressed during maturation. Functional annotation revealed coordinated enrichment in plastid biogenesis and protein import, lipid and cuticle remodeling, phenylpropanoid metabolism, transcriptional regulation, auxin-associated pathways, redox homeostasis, and membrane-associated transport. Refinement of this set defined an 81-gene SCC4-associated module and a 33-gene core cohort exhibiting pronounced early-stage dominance. Importantly, these transcriptional shifts preceded detectable induction of canonical C₄ enzyme genes, indicating that structural reorganization and plastid-associated processes are established before biochemical C₄ specialization. Collectively, our results delineate an early transcriptional program underlying SCC4 priming in a non-Kranz C₄ species.