The enhancement of grain chalkiness tolerance and weight by OsS40-2 mutation is linked to source-sink homeostasis in a developmental-dependent manner

Grain chalkiness is attributable to polygenic traits, including inadequate photosynthate supply, imbalanced source-sink dynamics, or restricted biosynthesis of storage materials. Nevertheless, the precise regulatory mechanism of chalkiness formation remains to be elucidated. In this study, a CRISPR/Cas9 gene editing mutant (oss40-2cr) of a novel transcription factor OsS40-2 in rice exhibited a longer and stay-green flag leaf, a lower CO2 assimilation rate, as well as a transparent endosperm, accompanying by augmented grain weight and diminished grain size compared to NIP. The complementation line oss40-2com partially restored the oss40-2cr phenotype to its NIP state. The integrative analysis of the transcriptome and the tsCUT&Tag dataset of the flag leaf and grain at varying developmental stages of the oss40-2cr relative to NIP demonstrated that OsS40-2 functions both as an activator and as a repressor for various downstream genes, thereby regulating enzyme activities related to catalysis, kinase and transferase in the carbohydrate and protein metabolic processes. OsS40-2 directly targeted and activated the transcription of the precursor of the RuBisCO large subunit 1 (OspreRBCL1) before pollination and that of the sucrose transporter OsSWEET7d before seed maturation. However, OsS40-2 exerts a contrary effect by repressing the transcription of OsUGT201 and OsGPIT1 gene at the seed development stage. OsS40-2 plays a critical node linking photosynthetic efficiency, grain filling and seed component metabolism through the maintenance of source-sink homeostasis in a developmental-dependent manner, thereby affecting grain weight and grain quality.