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Ribulose\u20101,5\u2010bisphosphate carboxylase\/oxygenase (Rubisco) a hetero\u2010oligomeric enzyme composed of chloroplast\u2010encoded large subunits (\n                          <jats:italic>rbcL<\/jats:italic>\n                          ) and nuclear\u2010encoded 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\n                          <jats:italic>rbcL<\/jats:italic>\n                          and\n                          <jats:italic>rbcS<\/jats:italic>\n                          sequences, expression profiles, and functional interactions across diploid progenitors (\n                          <jats:italic>Oryza<\/jats:italic>\n                          species bearing B and C genomes) and their derived allotetraploids.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          We performed Sanger sequencing of\n                          <jats:italic>rbcL<\/jats:italic>\n                          and\n                          <jats:italic>rbcS<\/jats:italic>\n                          coding regions across multiple accessions of B\u2010 and C\u2010genome diploids and corresponding allotetraploids. Sequence variation was characterized using nucleotide diversity (\u03c0), haplotype analysis, and dN\/dS ratio estimation to infer selective regimes. Gene conversion events were detected via phylogenetic incongruence and alignment\u2010based methods. Quantitative real\u2010time PCR (qRT\u2010PCR) was employed to assess tissue\u2010specific and developmental expression levels of\n                          <jats:italic>rbcS<\/jats:italic>\n                          paralogs and\n                          <jats:italic>rbcL<\/jats:italic>\n                          . Protein\u2013protein interaction affinities between\n                          <jats:italic>rbcL<\/jats:italic>\n                          and\n                          <jats:italic>rbcS<\/jats:italic>\n                          variants were systematically evaluated using yeast two\u2010hybrid (Y2H) assays under standardized conditions.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          Amino acid substitutions in\n                          <jats:italic>rbcL<\/jats:italic>\n                          specifically within the structural interface region known to mediate physical contact with\n                          <jats:italic>rbcS<\/jats:italic>\n                          were identified in both B\u2010 and C\u2010genome lineages. In contrast,\n                          <jats:italic>rbcS<\/jats:italic>\n                          homologs exhibited exceptionally high sequence conservation across all taxa, consistent with strong purifying selection (\u03c9 = dN\/dS \u226a\u20091); notably, no nonsynonymous single\u2010nucleotide polymorphisms (nsSNPs) were observed in\n                          <jats:italic>rbcS<\/jats:italic>\n                          coding sequences of the allotetraploids. Neither SNP distribution nor quantitative expression data revealed consistent parental\u2010biased allelic expression or dosage effects. However, Y2H assays demonstrated significantly stronger interaction intensities between maternally inherited\n                          <jats:italic>rbcL<\/jats:italic>\n                          alleles and paternally inherited rbcS alleles, suggesting preferential functional compatibility across parental origins.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          While no direct mechanistic linkage was established between cytonuclear coordination and maternal inheritance of\n                          <jats:italic>rbcS<\/jats:italic>\n                          , the pronounced evolutionary constraint on\n                          <jats:italic>rbcS<\/jats:italic>\n                          underscores its critical role in maintaining Rubisco functionality. The observed asymmetry in\n                          <jats:italic>rbcL<\/jats:italic>\n                          \u2013\n                          <jats:italic>rbcS<\/jats:italic>\n                          interaction preferences may reflect nascent coadaptation processes rather than fixed coevolutionary outcomes. Given the relatively recent origin of\n                          <jats:italic>Oryza<\/jats:italic>\n                          allotetraploids (estimated at &lt;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.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                    <\/jats:list>\n                  <\/jats:p>","DOI":"10.1111\/plb.70203","type":"journal-article","created":{"date-parts":[[2026,3,17]],"date-time":"2026-03-17T04:18:57Z","timestamp":1773721137000},"page":"1016-1028","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rubisco large and small subunits provide insights into allotetraploidy events from the genus\n                    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