Source-sink coordination serves as the foundation for improving crop yield. Current research primarily focuses on individual factors, such as increasing the source or expanding the sink, which often leads to disrupted source-sink balance, causing trade-offs among photosynthesis, yield, and stress response. To address these limitations, we present an integrated synthetic biological framework that synergistically enhances photosynthetic efficiency (source capacity), sink optimization, and abiotic stress tolerance. We developed an editing-overexpression coupling (EOC) vector system enabling simultaneous overexpression of four photosynthesis-enhancing genes (Cyt c₆, PsbA, FBPase, OsMGT3), knockout of three yield-limiting genes (GS3, Gn1a, OsAAP5), and self-excision of selection markers, gene-editing modules, and fragment deletion cassettes. Field evaluations of CFMP-gga transgenic lines revealed significant physiological improvements, including 13%–17% increase in photosynthetic rates, improved chlorophyll fluorescence parameters, and increased stomatal conductance. These enhancements translated into remarkable agronomic gains, including 18.7%–22.3% higher grain yield, 23.1%–26.1% increased biomass, and improved panicle architecture (increased grain size and grain number per panicle). The engineered lines maintained superior thermotolerance (under 42 °C stress) and alkali tolerance (at pH 10) compared to wild-type controls. This study provides a strategy for enhancing crop yield by demonstrating that coordinated multi-gene regulation of source-sink dynamics, coupled with stress resilience engineering, achieves concurrent improvements.

Plant anatomy is patterned early during leaf development which suggests studying the spatial–temporal transcriptomes of primordia will help identify critical regulative and functional genes. We successfully isolated the leaf primordia tissues from the C₃ grass rice and the C₄ grass foxtail millet by laser capture microdissection (LCM) and studied the gene expression throughout leaf developmental stages. Our data analysis uncovered the conserved expression patterns of certain gene clusters both in rice and foxtail millet during leaf development. We revealed genes and transcription factors involved in vein formation, stomatal development, and suberin accumulation. We identified 79 candidate genes associated with functional regulation of C₄ anatomy formation. Screening phenotype of the candidate genes revealed that knock-out of a putative polar auxin transport related gene NAL1 resulted significantly reduced veinal space in rice leaf. Our present work provides a foundation for future analyses of genes with novel functions in grasses and their role in leaf development, in particular the role in leaves with a contrasting C₃ vs. C₄ biosynthetic pathway.