T6P-mediated source-sink coordination regulates grain filling of rice inferior spikelets

Insufficient grain filling of inferior spikelets constrains yield and quality in large-panicle rice varieties. While source-sink coordination is crucial, its regulatory mechanisms, are not well understood. This study examined two rice varieties, JH809 and YY1540, to investigate grain-filling regulation. Despite YY1540 having lower pre-flowering assimilate reserves and leaf area per spikelet, its inferior spikelets demonstrated improved grain filling, increased starch accumulation, and enhanced grain quality compared to JH809. Dynamic analyses revealed that inferior spikelets of YY1540 transitioned into the active grain-filling period more rapidly, accompanied by stronger sink activity, as reflected by the rapid increase in starch-synthesis enzyme activities and ATP levels. In addition, source organs in YY1540 exhibited a more efficient response. To identify the key regulatory factors involved in the formation of sink activity, we conducted the treatment of removing superior spikelets to generate the material with accelerated and improved grain filling of inferior spikelets. Proteomic profiling of inferior spikelets transitioning from a stagnant to an active grain-filling state identified 5,081 differentially expressed proteins, with significant enrichment in pathways related to starch and sucrose metabolism and trehalose-6-phosphate (T6P) biosynthesis pathways. T6P, a sugar signaling molecule, exhibited a rapid rise-and-fall pattern, positively correlating with grain-filling rate in fast-filling grains, but showed a slow pattern with no significant correlation in slow-filling grains. Chemical intervention using the T6P precursor DMNB‑T6P demonstrated that early application (10 days after flowering) on inferior spikelets enhanced sink activity and concurrently stimulated source photosynthesis and assimilate remobilization, thereby improving grain‑filling efficiency. In contrast, late application (25 days after flowering) on inferior spikelets had limited promotive effects on sink activity but induced sugar accumulation in leaves, reduced photosynthesis, and accelerated leaf senescence, ultimately prematurely ending the grain filling process. Collectively, this study highlights the critical importance of rapid grain-filling progression in inferior spikelets and proposes a T6P-mediated, developmentally context-dependent model of source-sink coordination, providing new insights into improving grain-filling efficiency in inferior spikelets.