Multiple Proteins Interacting with VRN1 in Wheat

Wheat vernalization gene VRN1 was previously cloned according to a qualitative segregation pattern for vernalization requirement between winter wheat and spring wheat. Allelic variation in VRN1 was also associated with a quantitative trait locus controlling developmental trait segregation in winter wheat populations. When VRN1 in diploid wheat was completely deleted, the plant lost the ability to transition from vegetative to reproductive development, strictly maintaining a vegetative state. These previous studies combined suggest that signals from different flowering pathways may converge at VRN1 as an indispensable protein for phase transition in wheat. We used VRN1 as bait to search for its protein partners in a yeast two hybrid (Y2H) library, and several proteins were fished out. The in vitro protein pulldown assay was used to confirm a direct binding of VRN1 with SOC1 (T. aestivum wheat suppressor of overexpression of Constans 1) and VRT2 (T. aestivum wheat vegetative to reproductive transition-2). When VRN1 was used to test interactions with VRT2 and SOC1 in the same pulldown reaction, VRN1 showed greater interaction strength with VRT2 than with SOC1. These findings suggest that innate mechanisms exist which modulate competition of signal proteins from multiple pathways for VRN1 as a central regulator for flowering in wheat. The in vitro protein pulldown assay was also used to identify novel proteins which interact with VRN1 in wheat.