Chloroplast photorespiratory bypass in tomato couples carbon-nitrogen assimilation to increase yield and fruit...

Photorespiration is vital for C₃ plant carbon (C) and nitrogen (N) metabolism, yet most engineering ignores N-related constraints. Here, we engineered a chloroplast-targeted bypass in tomato (GCMG), comprising glycolate oxidase 1 (SlGLO1), catalase 2 (SlCAT2), malate synthase A (SlMSA), and glutamine synthetase 2 (SlGS2). This bypass integrates carbon concentration with enhanced ammonium reassimilation via the GS2/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. GCMG plants showed synergistic improvements in photosynthesis, biomass, and fruit quality; while total yield significantly increased over wild-type (WT), GCMG maintained a favorable trend beyond C-focused GCM lines. Mechanistically, GCMG partitions glycolate flux into parallel routes, maintaining N assimilation while enhancing chloroplastic CO₂ enrichment. ¹⁵N-labeling confirmed this metabolic synergy, revealing a 146% higher N turnover rate (f_new). GCMG also sustained robust N assimilation under elevated CO₂ and conferred resilience to N-deficiency, high oxygen, and heat stress. Coordinating C/N metabolism boosts productivity and resilience, offering a blueprint for crop improvement.