C₄ photosynthesis exemplifies convergent evolution of complex traits. Herein, we construct chromosome-scale genome assemblies and perform multi-omics analysis for five Flaveria species, which represent evolutionary stages from C₃ to C₄ photosynthesis. Chromosome-scale genome sequence analyses reveal a gradual increase in genome size during the evolution of C₄ photosynthesis attributed to the expansion of transposable elements. Systematic annotation of genes encoding C₄ enzymes and transporters identify additional copies of three C₄ enzyme genes through retrotranspositions in C₄ species. C₄ genes exhibit elevated mRNA and protein abundances, reduced protein-to-RNA ratios, and comparable translation efficiencies in C₄ species, highlighting a critical role of transcriptional regulation in C₄ evolution. Furthermore, we observe an increased abundance of ethylene response factor (ERF) transcription factors and cognate cis-regulatory elements associated with C₄ genes regulation. Altogether, our study provides valuable genomic resources for the Flaveria genus and sheds lights on evolutionary and regulatory mechanisms underlying C₄ photosynthesis.

Hybrid rice breeding has significantly increased the yield and stability of rice production. However, due to trait segregation in F2 progeny seeds, the F1 hybrid seeds must be renewed annually. Apomixis is a form of reproduction leading to clonal seeds, which has a revolutionary potential in fixing heterosis and realizing selfretention of hybrid rice seeds. Previously, we successfully engineered synthetic apomixis in rice by combining Mitosis instead of Meiosis (MiMe) with haploid induction (mutation of MATRILINEAL), a strategy known as Fix for Fixation of hybrids. However, only one hybrid rice variety has been tested for the Fix synthetic apomixis system. In this study, we expanded the application of the Fix strategy and achieved synthetic apomixis in multiple hybrid rice varieties. We observed significant variations in seed setting rate across diverse genetic backgrounds and witnessed a remarkable tenfold increase in clonal seed efficiency. Our findings will provide valuable insights into the application of the Fix strategy for synthetic apomixis in hybrid rice.

Background/Objectives: BABY BOOM (BBM), a transcription factor from the APETALA2 (AP2) protein family, plays a critical role in somatic embryo induction and apomixis. BBM has now been widely applied to induce apomixis or enhance plant transformation and regeneration efficiency through overexpression or ectopic expression. However, the structural and functional evolutionary history of BBM genes in plants is still not well understood. Methods: The protein sequences of 10 selected plant species were used to locate the branch of BBM-Like by key domain identification and phylogenetic tree construction. The identified BBML genes were used for further conserved motif identification, gene structural analysis, miRNA binding site prediction, cis-acting element prediction, collinear analysis, protein–protein interaction network construction, three-dimensional structure modeling, molecular docking, and expression pattern analysis. Results: A total of 24 BBML proteins were identified from 10 representative plant species. Phylogenetic relationship analysis displayed that BBML proteins from eudicots and monocots were divided into two clusters, with monocots exhibiting a higher number of BBMLs. Gene duplication events indicated that whole genome/segmental duplication were the primary drivers of BBML genes’ evolution in the tested species, with purifying selection playing a key role during evolution processes. Comparative analysis of motif, domains, and gene structures revealed that most BBMLs were highly evolutionarily conserved. The expression patterns of BBML genes revealed significant tissue specificity, particularly in the root and embryo. We also constructed protein–protein interaction networks and molecular docking models to identify functional pathways and key amino acid residues of BBML proteins. The functions of BBMLs may differ between monocots and eudicots, as suggested by the functional enrichment of interacting proteins. Conclusions: Our research delved into the molecular mechanism, evolutionary relationships, functional differentiation, and expression patterns of BBML genes across plants, laying the groundwork for further investigations into the molecular properties and biological roles of BBMLs.

C₄ photosynthesis represents a remarkable example of convergent evolution of complex traits. In this study, we present an integrated genomic analysis of the genus Flaveria, which includes species at different evolutionary stages from C₃ to C₄ photosynthesis. The chromosome-scale genome sequences of five Flaveria species, representing C₃, intermediate, and C₄ types, reveal a gradual increase in genome size attributed to the expansion of transposable elements (TEs). Through a combination of gene phylogeny and transcript abundance analysis, we systematically identified genes encoding C4 enzymes and transporters. We further found that C₄ species acquired additional copies of genes encoding three C4 enzymes through retrotranspositions. Moreover, we observed an increased abundance of ERF cis-regulatory elements and ERF transcriptional factors associated with the regulation of C₄ genes in C₄ species compared to non-C₄ species. Notably, C₄ genes exhibited elevated both RNA and protein levels but decreased protein-to-RNA ratios in C₄ species. Additionally, codon usage patterns are conserved among the five Flaveria species. All these results suggest a predominant role of transcriptional regulation in the evolution of C₄ photosynthesis. Our comprehensive dataset also provides valuable resources to study the evolution and regulatory mechanisms underlying C₄ photosynthesis.

The genus Flaveria, containing species at different evolutionary stages of the progression from C3 to C4 photosynthesis, is used as a model system to study the evolution of C4 photosynthesis. Here, we report chromosome-scale genome sequences for five Flaveria species, including C3, C4, and intermediate species. Our analyses revealed that both acquiring additional gene copies and recruiting ethylene responsive factor (ERF) cis-regulatory elements (CREs) contributed to the emergence of C4 photosynthesis. ERF transcriptional factors (TFs), especially intronless ERF TFs, were co-opted to regulate C4 photosynthetic genes in both dicotyledonous and monocotyledonous C4 species that diverged 160 million years ago (mya). These C4 species co-opted intronless ERF TFs originated from the Late Ordovician mass extinction that occurred ~450 mya in coping with environmental stress. Moreover, intronless ERF TFs are the most abundant intronless TFs in plant genomes, and show fast responses to environmental changes. Therefore, this study suggested that intronless ERF TFs, acquired during the early evolution of plants, provided the molecular toolbox facilitating multiple subsequent independent evolutions of C4 photosynthesis.