Identification and Expression Analyses of Genes Involved in Early Endosperm Development in Arabidopsis and Cereals

Cereal seeds are important source for food, feed and also provide an industrial raw material. A sound understanding of seed initiation and the control of early endosperm development will be important for increasing cereal crop yield and improving grain quality using biotechnology. The approach taken in this project was to investigate the potential application of candidate genes and pathways for manipulating endosperm initiation and development. The candidate system chosen was the FIS class Polycomb group complex since there has been considerable work on the role of this complex in the transcriptional regulation of seed initiation and endosperm cellularization in Arabidopsis. The aims of the work described in this thesis were, first to identify and characterize novel proteins which directly interact with the known PcG members FIE and SWN. These two proteins play a key role in the regulation of endosperm proliferation in Arabidopsis. The second aim was to identify homologs from rice and Arabidopsis which potentially regulate grain size, weight and composition and to investigate the expression profiles and possible functions of these genes and isolate endosperm-specific promoters. Several novel plant PcG complex proteins from Arabidopsis were identified via yeast two-hybrid (Y2H) screens. Several other interacting transcription factors of FIE were identified, including SWN (a.k.a. EZA1/SWINGER), AREB2 (ABA-responsive element binding factor 2), VPL (Viviparous-like) and ICE2 (Inducer of CBF Expression 2). Interacting partners of SWN were also isolated. These included AtRING-H2, an E3 type protein ubiquitin ligase, v-SNARE, and BrD (Bromodomain-containing protein), and a plant PHD finger-containing factor. The expression profile of these genes was studied using quantitative real-time PCR (qRT-PCR) in a series of Arabidopsis tissues prepared from wild type (WT) plants. Expression of these genes was also studied by using the predicted 5'-regulatory sequences of SWN, AtRING-H2, AREB2, VPL and ICE2 fused to GUS reporter gene and stably transformed into Arabidopsis. The expression pattern of each candidate was revealed by GUS assays. Results showed that the VPL promoter was specific to the male gamete, while the AtRING-H2 promoter was found to be male gametophyte- and seed-specific. In order to elucidate the functions of the key PcG partner SWN and its possible role in the regulation of seed initiation, T-DNA insertion lines were analyzed and gene knockdown constructs were developed. Seed development was disrupted by 50% in one SWN silencing line via dsRNAi. A preliminary study of the SWN silencing plant was carried out but further characterization is needed to uncover the role of SWN in seed development. Manipulation of endosperm development will be dependent on access to promoters specific to early endosperm development. Arabidopsis END1, a homolog of a barley endosperm marker gene was characterized in this project. AtEND1 was identified as a member from Arabidopsis lipid transfer protein family. This gene was specifically expressed in gametophytes and developing seeds during free nuclear division and endosperm cellularization. A number of seed-specific cis-acting elements were identified in the 5'-regulatory region of AtEND1. The promoter-GUS construct was also transformed into canola. A less specific expression pattern was observed in transgenic canola plants. Analyses of the transgenic plants in Arabidopsis and canola are presented. Three rice orthologs at END1 (OsPR602, OsPR9a and OsPRPI) were identified from database searches. The expression of these genes in rice was analyzed by qRT-PCR. Transcriptional GUS fusion constructs of the predicted promoter regions were transformed into rice and barley. Analyses of transgenic barley and rice plants were performed by using PCR screening, Southern blot hybridization and histological GUS assay. These genes were predominantly expressed in the developing rice endosperm. OsPR602 and OsPR9a showed similar spatial and temporal expression patterns in barley compared to their expressions in rice. Expression analysis found that OsPRPI directed gene expression in the vascular bundles of anthers, hulls and micropylar and chalazal poles of pistil shortly before and after anthesis and the vascular bundles of developing caryopses until 19 days after pollination in rice. There was no GUS expression in barley under the control of OsPRPI. The studies also revealed wounding-induced expression of this gene in the vascular traces of leaf blade, leaf sheath, stem and grains. The induction and expression level of OsPRPI was studied by northern blot analysis and qRT-PCR. These gene promoters will be useful tools in transgene research and in the manipulation of cereal grain yield, quality and tolerance to biotic and abiotic stresses.