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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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