The OsbHLH166-OsABCB4 module regulates grain length and weight via altering auxin efflux

Grain size is one of the key factors determining grain weight and yield. However, it remains largely unknown how the auxin signal regulates grain size in rice. Here, a quantitative trait locus qGL1 for grain length was identified with recombinant inbred lines 9311 and Nipponbare (NIP), and fine-mapped to the OsABCB4 gene, which encodes a member of the ATP Binding Cassette B (ABCB) subfamily. Compared to NIP, loss of OsABCB4 function leads to longer and heavier grains, while over-expression of OsABCB4 causes shorter and lighter grains, demonstrating a negative control of grain length and weight in rice. Haplotype analyses using 3024 rice-sequenced genomes indicated that SNP₂₅ and SNP₅₅ in OsABCB4 are correlated with grain length in rice. OsbHLH166, a basic helix-loop-helix transcription factor, was screened from a yeast one-hybrid library and confirmed to directly bind the promoter of OsABCB4 by electrophoretic mobility shift assay and yeast one hybridization validation and to activate the expression of OsABCB4 by dual-luciferase reporter assay. And OsbHLH166 expressions matching OsABCB4 were all higher expressed in stems and glumes. OsABCB4 was subcellularly localized on the plasma membrane, which was verified as an auxin efflux transporter. Histological analysis, IAA content measurement and DR5:GUS activity analyses demonstrated that OsbHLH166 and OsABCB4 control grain length by regulating cell expansion and auxin contents in glume cells and altering the expression of GL7, GS2, TGW3 and GS3 genes during rice grain development. This newly identified OsbHLH166-OsABCB4 module sheds light on our understanding of molecular mechanisms underlying the regulation of grain size and weight via auxin and provides a new gene resource for molecular design breeding of grain type.