Samuel Menge

Seed size regulation in Arabidopsis plants is controlled by a number of signaling pathways that each have their own unique mechanisms of interaction. The IKU2-MINI3-SHB1 signaling pathway is one such method that is critical to embryonic growth and development within seeds of angiosperms in terms of endosperm tissue proliferation. Increased proliferation of endosperm early in the Arabidopsis life cycle leads to larger seed cavities which are able to facilitate future embryonic growth within larger seeds. Furthermore, the stored nutrients within the endosperm are important in supporting future embryonic growth and development. The nuclear protein SHB1 and the transcription factor MINI3 are endosperm proliferation proteins that are recruited to the promoter of the IKU2 gene to activate it, producing a leucine-rich repeat kinase that acts as one of the key regulators of seed size in Arabidopsis plants. However, only MINI3 can directly bind to the IKU2 promoter and SHB1 is indirectly bound to MINI3, so the mechanism of how SHB1 affects IKU2 expression and other possible proteins involved in the signaling pathway are still unknown. Here we show that the Arabidopsis Response Regulator 11 (ARR11) protein likely serves as the primary intermediary protein between MINI3 and SHB1 to allow them to signal each other to activate IKU2 expression. We found in controlled in vitro Yeast Two-Hybrid (Y2H) assays that ARR11 displays a strong interaction with MINI3 as characterized by the ONPG assays of the experimental Y2H vectors producing 42.216 galactosidase units, nearly double that of the empty vector control. When mapping the C-/N-terminal domains of ARR11 interaction with MINI3 and vice versa, we found that the C-terminus of ARR11 is the likely binding site with MINI3 and that the entire MINI3 protein is necessary for binding with ARR11. Furthermore, we found that ARR10 and ARR18 display strong interactions with each other but not with ARR11 suggesting that they may not be as involved in IKU2 expression as originally proposed. Overall, our results demonstrate that the ARR protein family plays a key role in activation of the IKU2 seed size regulator gene and the expression of its kinase which is critical for endosperm proliferation in Arabidopsis. We anticipate our assays will provide a starting point for genetic modification targets within Arabidopsis that, when stimulated to higher levels of expression, will likely lead to higher quantities of endosperm proliferation and a larger seed cavity in offspring. For example, in vivo experiments testing overexpression of the IKU2 pathway protein genes could be done to see the effects on seed size. Furthermore, there are many seed-production plants that have isolated gene homologues similar to IKU2 that could be studied by looking for similar transcription factors and nuclear proteins that may lead to larger seed cavities when overexpressed as well.