Graduate Thesis Or Dissertation
 

Study of seed germination-associated genes using Arabidopsis enhancer-trap

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  • In mature Arabidopsis seeds, the testa (seed coat) is no longer a living tissue. Thus, major sites of gene expression in imbibed seeds are the internal living tissues such as the embryo and the endosperm. To elucidate the molecular mechanisms of seed germination, the gene regulation in these two tissues during germination needs to be investigated. An enhancer-trap approach was used in this study to identify tissue- and stage-specific gene expression in seeds. The Arabidopsis enhancer-trap lines (Thomas Jack’s population, CS31086) from the Arabidopsis Biological Resource Center (ABRC) were screened for β-glucuronidase (GUS) expression in imbibed seeds in order to identify and characterize seed germination-associated genes. One hundred twenty one independent lines exhibiting diverse tissuespecific GUS expression patterns were isolated and kept as the Seed-GUSExpression enhancer-trap library. This library was donated to the ABRC (stock no. CS24362–CS24480), and is now available to the international seed research community. Ninety one lines showed GUS expression predominantly in the micropylar end of the seed (named BME [Blue Micropylar End] lines), indicating that the micropylar region of Arabidopsis seed is selectively activated during germination. One of these lines, BME3, had a T-DNA insertion site in the 5’ upstream region of a GATA-type zinc finger transcription factor gene (termed BME3-ZF). The BME3-ZF mRNA accumulated in seeds during cold stratification, suggesting its involvement in the physiological changes that accelerate the release of dormancy. BME3-ZF was expressed just prior to the expression of two GA biosynthesis genes, AtGA20ox3 and AtGA3ox1 which are also induced by cold stratification. The BME3-ZF knockout plants produced seeds exhibiting increased dormancy, which showed reduced response to cold stratification. The ungerminated knockout seeds exhibited testa rupture, but failed to penetrate the endosperm layer. Application of gibberellic acid (GA₃) rescued impaired germination of the knockout seeds without cold stratification, indicating that the normal GA signal transduction pathway is present in the knockout seeds. These results indicate BME3 GATA zinc finger protein is a positive regulator of Arabidopsis seed germination. In this study, we provide the proof-of-concept study for the Seed-GUS-Expression enhancer trap library and the detailed procedures to use a combination of gene-expression analysis of wild-type seeds and functional analysis using knockout plants for encouraging international collaborations to utilize this library which is a useful tool to identify the genes critical for seed germination.
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file details Po-Pu_Liu-PhD_dissertation-3-27-07.pdf 2017-10-20 Público Baixar