Rubisco large and small subunits provide insights into allotetraploidy events from the genus Oryza

Cytonuclear interactions play a pivotal role in eukaryotic adaptation, particularly in polyploid organisms, which must achieve coordinated expression and functional compatibility between nuclear-encoded and cytoplasmically inherited (e.g., chloroplast- and mitochondrial-encoded) genes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) a hetero-oligomeric enzyme composed of chloroplast-encoded large subunits (rbcL) and nuclear-encoded small subunits (rbcS) provides an ideal model system for investigating the evolutionary dynamics of cytonuclear coadaptation. To elucidate patterns of cytonuclear evolution following polyploidization, we comparatively analysed rbcL and rbcS sequences, expression profiles, and functional interactions across diploid progenitors (Oryza species bearing B and C genomes) and their derived allotetraploids. We performed Sanger sequencing of rbcL and rbcS coding regions across multiple accessions of B- and C-genome diploids and corresponding allotetraploids. Sequence variation was characterized using nucleotide diversity (π), haplotype analysis, and dN/dS ratio estimation to infer selective regimes. Gene conversion events were detected via phylogenetic incongruence and alignment-based methods. Quantitative real-time PCR (qRT-PCR) was employed to assess tissue-specific and developmental expression levels of rbcS paralogs and rbcL. Protein-protein interaction affinities between rbcL and rbcS variants were systematically evaluated using yeast two-hybrid (Y2H) assays under standardized conditions. Amino acid substitutions in rbcL specifically within the structural interface region known to mediate physical contact with rbcS were identified in both B- and C-genome lineages. In contrast, rbcS homologs exhibited exceptionally high sequence conservation across all taxa, consistent with strong purifying selection (ω = dN/dS ≪ 1); notably, no nonsynonymous single-nucleotide polymorphisms (nsSNPs) were observed in rbcS coding sequences of the allotetraploids. Neither SNP distribution nor quantitative expression data revealed consistent parental-biased allelic expression or dosage effects. However, Y2H assays demonstrated significantly stronger interaction intensities between maternally inherited rbcL alleles and paternally inherited rbcS alleles, suggesting preferential functional compatibility across parental origins. While no direct mechanistic linkage was established between cytonuclear coordination and maternal inheritance of rbcS, the pronounced evolutionary constraint on rbcS underscores its critical role in maintaining Rubisco functionality. The observed asymmetry in rbcL-rbcS interaction preferences may reflect nascent coadaptation processes rather than fixed coevolutionary outcomes. Given the relatively recent origin of Oryza allotetraploids (estimated at <0.5 million years), ongoing cytonuclear adjustment potentially mediated by regulatory rewiring, subfunctionalization or epigenetic modulation likely contributes to the stabilization of photosynthetic machinery during early polyploid evolution.