Abstract: Seed size is a critical factor in determining crop yield. Recent studies highlighted the importance of the MPK3/MPK6 cascade in regulating seed size in both Arabidopsis and rice. However, the underlying mechanism is not fully elucidated. Here, we report a novel function for the MKK4/MKK5–MPK3/MPK6 module in controlling the timing of endosperm cellularization, thereby regulating the seed size. Loss of function of MKK4/MKK5 or MPK3/MPK6 results in precocious endosperm cellularization and smaller seeds. WRKY transcription factor WRKY10/MINI3 is identified as a substrate of MPK3/MPK6 in this process. During early silique development, MINI3 is expressed and undergoes phosphorylation, a process mediated by MPK3/MPK6 at five specific serine residues. The phospho-deficient MINI3^SA variant loses the function of wild-type MINI3 in vivo. Furthermore, we found that IKU2 and RLK7, two closely related receptor-like kinases, function upstream of the MKK4/MKK5–MPK3/MPK6–MINI3 pathway to regulate seed size. The phosphorylation of MINI3 mediated by MPK3/MPK6 enhances its transcriptional activity toward target genes, including IKU2, RLK7, and MINI3 itself, thereby forming a positive regulatory loop within this signaling pathway. In summary, our study elucidates a signaling pathway composed of IKU2/RLK7–MAPKKK(s)–MKK4/MKK5–MPK3/MPK6–MINI3 that regulates endosperm cellularization and seed size, providing a theoretical basis for crop breeding.

Key words: endosperm cellularization, IKU2, MAPK cascade,  MINI3, RLK7, seed size