Proposal number: IOS-0919886 Proposal Title: "SHB1 regulates seed development in Arabidopsis"
Double fertilization in angiosperms leads to the formation of a diploid embryo and a triploid endosperm. In Arabidopsis, seed development undergoes an initial phase of active endosperm proliferation followed by a second phase in which embryo grows rapidly at the expense of the endosperm. Since mature seed size is largely attained during the initial phase, seed size is coordinately regulated by the growth of the maternal ovule, the endosperm, and the embryo. The mechanisms underlying seed size control are still not well understood, yet seeds form the bulk of the diet of human population. The gene SHB1 is a positive regulator of Arabidopsis seed development that operates through regulation of both cell size and cell number. shb1-D, a gain-of-function allele, increases seed size and shb1, a loss-of-function allele, reduces seed size. The increase in seed size of shb1-D is associated with the timing of endosperm cellularization, enlargement of chalazal endosperm, and subsequent embryo development. SHB1 regulates the expression of the genes MINI3 and IKU2, which encode a WRKY transcription factor and an LRR receptor kinase respectively. SHB1 associates with MINI3 and IKU2 promoters in vivo, and directly interacts with MINI3. The goal of this project is to test if SHB1 is recruited as a co-activator by a WRKY transcription factor to control gene expression required for endosperm development. Various cellular, biochemical, and molecular approaches will be used to investigate the interaction of SHB1 with MINI3 and the biological consequence of SHB1-MINI3 interaction. Understanding the mechanisms of seed size regulation has the potential to generate a potential increase in seed yield and a concomitant increase in the amount of protein and oil produced per seed.
This project will provide excellent opportunities for undergraduates, graduate students, and postdoctoral fellows to gain significant research experiences, and most importantly, to develop their ability to critically analyze and solve scientific problems. Seed size is a yield trait that traditional plant breeding has limited success in improving. Seed development in major seed crops such as soybean and canola follows a very similar path to Arabidopsis. Enhancing the potential for large seed size represents one of the most promising and least explored avenues for generating a significant increase in agricultural yield.
SHB1 is a positive regulator of Arabidopsis seed development. SHB1 over-expression significantly affects endosperm proliferation, timing of endosperm cellularization, and embryo development. At maturity, seeds of SHB1 over-expression lines have more cells and larger cells. SHB1 directly regulates the expression of two genes, MINI3 and IKU2, a WRKY transcription factor gene and an LRR receptor kinase gene. Mutation in either MINI3 or IKU2 affects endosperm development and reduces seed size. With this NSF support, we investigate how, when, and where SHB1 regulates the expression of MINI3 and IKU2 at different days after pollination in either gain-of-function or loss-of-function SHB1 mutants. SHB1 directly associates with the MINI3 or the IKU2 promoter, and this association requires MINI3. Similar to SHB1, MINI3 also associates with the same regions of its own and the IKU2 promoter, and interacts with SHB1. In a vivo trans-activation assay, MINI3 is delivered to mini3/shb1, mini3/SHB1, and mini3/shb1-D backgrounds along with MINI3 promoter-GUS or IKU2 promoter-GUS reporter. MINI3 barely activates transcription from pMINI3::GUS or pIKU2::GUS, but SHB1 strongly activates the transcription from the promoter-reporter constructs. The mechanisms underlying the control of seed size are still not well understood, and the proposed activities will significantly advance our current knowledge on seed size regulation. Seed size is the yield trait that traditional breeding has had the most limited success in improving effectively, yet seeds form the bulk of the diet of human population. Seed development in major seed crops such as soybean and canola follows a very similar path as Arabidopsis. Enhancing the potential for large seed sizes represents one of the most promising and less explored avenues for significant increases in agricultural yields. We have integrated the basic scientific research in physiology, genetics, and molecular biology with the training of one postdoctoral associate, one graduate student, and several undergraduates over the grant period from 2009 to 2012. The postdoctoral associate and graduate student have been mentored in areas of career development and awareness, teaching and presentation effectiveness, communication and interpersonal skills, and professional experiences. This NSF support also sustained our efforts to train undergraduate lab assistants during regular semesters and to recruit a few undergraduates in the summer months from research-limited institutions such as Luther College and Truman State University. My laboratory has served as a harbor for a summer undergraduate research program supported by NSF at the University of Minnesota. I have primarily served as a research advisor, interacted closely with the students of female and color, and provided career development guides.
