Carbon isotope signature decodes the synergistic effect of flag leaf and ear in providing assimilates for grain-filling in wheat under tissue-specific light exclusion

• Spike-localized photosynthesis sustains wheat grain filling efficiency. • Thermotolerant cvs. sustain higher sugar and enzyme activity. • Light exclusion alters assimilate partitioning and nutrient flow. • Rubisco, SuSy, SOD genes are highly light-responsive in spikes. • Shade-resilient genotypes display enhanced source–sink plasticity. This study comprehensively investigated the influence of tissue-specific light exclusion on carbon metabolism, assimilate partitioning, and grain quality across four contrasting wheat genotypes (HD2967, HD3086, HD2329, PBW343). Employing ¹⁴C labeling, biochemical assays, and gene expression analysis, we quantified CO₂ fixation, sucrose synthase (SuSy), superoxide dismutase (SOD), soluble starch synthase (SSS) activities, and sugar, protein, and micronutrient accumulation in flag leaf, stem, and spike tissues. Under full light, HD2967 and HD3086 consistently exhibited superior photosynthetic efficiency and assimilate partitioning. Flag leaf shading significantly reduced CO₂ fixation and associated metabolic activities, confirming its pivotal role. Direct spike shading severely impaired its CO₂ fixation, significantly reduced its SuSy and SSS activities, and diminished grain N, Fe, and Zn accumulation, highlighting the spike's critical photosynthetic role in grain-filling and quality. Interestingly, flag leaf and stem often showed compensatory increases in reducing sugars when the spike was shaded, suggesting altered carbon partitioning. Significant genotypic variability was observed with HD2967 showed strong stem sink activity and efficient assimilate translocation. HD2329 exhibited remarkable metabolic flexibility and robust compensatory translocation, indicating superior shade resilience. PBW343 was highly spike-dependent and sensitive to light exclusion. Gene expression of RuBisCo, SuSy, sHSP17 , and SOD was light-dependent, with spike tissues displaying significant light responsiveness. These findings highlight tissue-specific light perception, particularly in the spike, for sustaining grain development and nutrient accumulation, offering insights for breeding climate-resilient wheat cultivars.