Non-catalytic and catalytic TREHALOSE-6-PHOSPHATE SYNTHASES interact with RAMOSA3 to control maize development

Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.