| Sexual reproduction is important for propagation and requires a series of cell division and differentiation events. In flowering plants, it is incompletely understood how these events are genetically controlled. Because transcriptional and post transcriptional regulations are suggested to be main molecular mechanisms in these events, it is important to examine the changes of all transcripts (transcriptome) and proteins (proteome) during plant reproductive development. However, only limited studies have been done to analyze the transcriptome and proteome during plant reproductive development. Part of my thesis work is an analysis of the transcriptome in the tapetum, which is an important sporophytic tissue that contributes to microsporogenesis and pollen formation. It is known that DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORES (AMS), two bHLH transcription factors, are required for the development and functions of the tapetum. However, it is poorly understood which genes are regulated by DYT1 and/or AMS. In Chapter 2, a transcriptome analysis of the dyt1-1 and ams mutants by using microarray is presented. It was found that DYT1 positively and negatively regulates approximately 600 and 300 genes, respectively, and that AMS positively and negatively regulates ∼600 and ∼900 genes, respectively. DYT1 is required to control a broad spectrum of tapetum functions in part by activating at least 32 transcription factors including AMS. However, AMS regulates not only common but also different sets of genes compared to DYT1, which suggests AMS might have distinct functions. In order to differentiate the direct and indirect regulation by DYT1, I searched the binding sites of DYT1 by in vitro (Selex and gel-shift) and in vivo (ChIP) protein-DNA interaction experiments, which is described in Chapter 3. The results indicate that DYT1 recognizes the E-box (CANNTG) in vitro and binds to its own promoter and the promoters of downstream genes in vivo. These findings not only reveal the spectrum of functions regulated by DYT1 but also suggest some mechanisms of the transcriptional controls in the tapetum. In Chapter 4, I present a systematic exploration of Arabidopsis floral proteome where I identify more than 2400 floral proteins and provide pieces of evidence for protein modifications (methylation, acetylation, and glycosylation etc.) of some proteins by using 2-Dimensional Gel Electrophoresis/mass spectrometry and Multidimensional Protein Identification Technique (MudPIT). These studies have provided new insights into transcriptional and post transcriptional regulation in plant reproductive development. |
