Bundle sheath cell-specific expression of chloroplast genes encoding subunits of the NADH dehydrogenase-like complex...

C₄ photosynthesis alleviates the limitation caused by the oxygenase activity of Rubisco by partitioning photosynthetic functions between two distinct cell types: bundle sheath cells (BSCs) and mesophyll cells (MCs). These cell types perform different steps of photosynthesis using specialized machinery, accompanied by differential expression of chloroplast genes. To uncover the underlying molecular mechanisms for this differentiation, we isolated BSCs and MCs and compared their chloroplast transcriptomes, focusing on the chloroplast NADH dehydrogenase-like (NDH) complex, which is enriched in BSCs. To investigate whether RNA stabilization contributes to differential gene expression, we analyzed RNA footprints that reflect the binding of pentatricopeptide repeat (PPR) proteins to their RNA targets. We could not detect cell-type-specific accumulation of footprint RNAs. We then focused on transcriptional regulation, specifically on an operon that starts with the rps15 gene. The operon includes six ndh genes and the psaC gene encoding a photosystem I subunit. Transcript levels of all genes in this operon were higher in BSCs than in MCs, suggesting coordinated regulation as a transcriptional unit. Based on the genomic location of the rps15 gene within inverted repeats near the junctions on both sides of the small single copy region, we demonstrated that rps15, through two distinct promoters, is sufficient to drive preferential accumulation of downstream transcripts in BSCs.