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

Graduation date: 2007In mature Arabidopsis seeds, the testa (seed coat) is no longer a living tissue. Thus,\ud major sites of gene expression in imbibed seeds are the internal living tissues such\ud as the embryo and the endosperm. To elucidate the molecular mechanisms of seed\ud germination, the gene regulation in these two tissues during germination needs to\ud be investigated. An enhancer-trap approach was used in this study to identify\ud tissue- and stage-specific gene expression in seeds. The Arabidopsis enhancer-trap\ud lines (Thomas Jack’s population, CS31086) from the Arabidopsis Biological\ud Resource Center (ABRC) were screened for β-glucuronidase (GUS) expression in\ud imbibed seeds in order to identify and characterize seed germination-associated\ud genes. One hundred twenty one independent lines exhibiting diverse tissuespecific\ud GUS expression patterns were isolated and kept as the Seed-GUSExpression\ud enhancer-trap library. This library was donated to the ABRC (stock no.\ud CS24362–CS24480), and is now available to the international seed research\ud community. Ninety one lines showed GUS expression predominantly in the\ud micropylar end of the seed (named BME [Blue Micropylar End] lines), indicating\ud that the micropylar region of Arabidopsis seed is selectively activated during\ud germination. One of these lines, BME3, had a T-DNA insertion site in the 5’\ud upstream region of a GATA-type zinc finger transcription factor gene (termed\ud BME3-ZF). The BME3-ZF mRNA accumulated in seeds during cold stratification,\ud suggesting its involvement in the physiological changes that accelerate the release\ud of dormancy. BME3-ZF was expressed just prior to the expression of two GA\ud biosynthesis genes, AtGA20ox3 and AtGA3ox1 which are also induced by cold\ud stratification. The BME3-ZF knockout plants produced seeds exhibiting increased\ud dormancy, which showed reduced response to cold stratification. The\ud ungerminated knockout seeds exhibited testa rupture, but failed to penetrate the\ud endosperm layer. Application of gibberellic acid (GA₃) rescued impaired\ud germination of the knockout seeds without cold stratification, indicating that the\ud normal GA signal transduction pathway is present in the knockout seeds. These\ud results indicate BME3 GATA zinc finger protein is a positive regulator of\ud Arabidopsis seed germination. In this study, we provide the proof-of-concept study\ud for the Seed-GUS-Expression enhancer trap library and the detailed procedures to\ud use a combination of gene-expression analysis of wild-type seeds and functional\ud analysis using knockout plants for encouraging international collaborations to\ud utilize this library which is a useful tool to identify the genes critical for seed\ud germination