GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.