F Objectives: Define where MLP1 gene is expressed and if MLP1 peptides are mobile? Assess if MLP1 regulates PIN1 localization and in turn embryo development? The endosperm is a seed tissue that surrounds and nourishes the embryo in flowering plants, much like how the placenta nourishes a mammalian embryo [1]. The endosperm (Fig. 1A) contains a balance of starches, oils and proteins that supports embryo development and likely control gene expression patterns in embryonic cells. As a result, mutation or loss of constituents from the endosperm can impact greatly on the embryo, restricting its growth and even viability. Figure 1. Cross section of an early developing maize seed (A) and schematic diagram showing the establishment of the apical-basal axis in a zygote (B). Green lines highlight expression of PIN1 auxin efflux carrier, which concentrates at the embryo apex and leads to increased auxin accumulation. This helps the embryo define its apical-basal orientation. One of the most important developmental landmarks of an embryo is the establishment of an apical-basal axis [2]. The plant hormone auxin mediates this process by accumulating at the apical region of the embryo. Auxin gradients are created by a trans-membrane protein channel known as PIN1 [3]. PIN1 expression (Fig 1B) is concentrated in the embryo apex and corresponds to auxin distribution, however, it is not yet known what mechanism directs this early pattering event. One recent hypothesis is that cysteine rich, small, extra- embryonic proteins control expression and localisation of PIN1 in the embryo. Wild type A188 maize Extra-Embryonic Proteins Regulate Embryo Development Student: Shannon Easterlow Supervised by Jose Gutiérrez-Marcos and Perry Bateman. School of Life Sciences. Jose Gutiérrez-Marcos and Perry Bateman for supervision. URSS for financial support Results: Summary: Experimental Design: Generation of mlp1 mutants in A188 maize Collect seeds at different Days After Pollination (DAP); 0, 2, 4, 6, 8, 10 and 12 DAP for comparative histological Analysis Immunolocalisation of MLP1 using antibodies conjugated to Alkaline Phosphatase (AP) Immunolocalisation of PIN1 using FITC fluorescent antibodies MLP1:GUS transcriptional reporter to visualise gene expression Introduction: Aim: Determine whether the cysteine-rich peptide MLP1 peptides regulates PIN1 expression in the embryo and thus its development. A B Figure 3. Immunolocalisation showing cellular localisation of MLP1 protein in the endosperm (in blue-purple) at different development stages of wild type maize. A-D are 0DAP, 2 DAP, 4DAP and 6DAP, respectively. (Pe) Pericarp, (se) starchy endosperm, (ESR) Embryo Surrounding Region, (e) embryo and (cc) central cell. Scale bars are 1mm. A D C B Figure 2. MLP1 expression analysis in developing seeds using a MLP1:GUS transcriptional reporter. Unfertilised ovules (A and B) and 10DAP seeds (C and D). (Nc) Nucellus, (cc) central cell, (es) embryo sac, (se) starchy endosperm, (ESR) Embryo Surrounding Region, (e) embryo and (cc) central cell. Scale bar 100µm. DC AB Future Work: Analyse the seed and embryo abnormalities found in MLP1 mutant seeds. Optimise the immunolocalisation of PIN1 and MLP1 in early developing maize seeds. Establish if MLP1 affects directly or indirectly auxin distribution in the developing embryo. MLP1 is exclusively expressed in the embryo sac and the endosperm of maize seeds. MLP1 peptides accumulate in cells surrounding the egg cell gamete and the developing embryo. PIN1 protein is highly abundant in the embryo surrounding region (ESR) and the basal endosperm transfer layer (BETL) of the maize endosperm, as well as in the apical portion of the embryo. MLP1 mutant seeds display abnormalities in embryo development associated with defects in the expression of PIN1. Figure 5. Sub-cellular localisation of PIN1 in 6DAP wild type (A) and MLP1 mutant seeds (B). Cell walls are stained with Calcofluor (green) and PIN1 is detected with a fluorescent labelled antibody (red). Figure 4. Immunolocalisation showing the cellular localisation of PIN1 (blue-purple stain) in 6DAP wild type (A) and MLP1 mutant seeds (B). B A B A e su ESR e su ESR e su ESR BETL e su ESR BETL References: [1] Regulation and Flexibility of Genomic Imprinting during Seed Development. The Plant Cell Michael T. Raissig, Célia Baroux, and Ueli Grossniklaus [2] Auxin and cytokinin regulate meristem formation in early embryogenesis. Molecular Plant Ying-Hua Su, Yu-Bo Liu, and Xian-Sheng Zhang [3] PIN1 auxin efflux carriers localisation in Zea mays. Landes Biosciences. C. Forestan Acknowledgements: