Chemically inducible gene expression in seeds before testa rupture

Impermeability of the testa hinders efficient penetration of some small chemicals, such as transcriptional inhibitors, through the endosperm and the embryo during seed experiments. In Arabidopsis seeds, 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid, a substrate for β-glucuronidase, did not permeate through the endosperm and embryo efficiently at the stages before testa rupture. The Arabidopsis testa also limited efficient entry of methoxyfenozide, a chemical ligand that was used for inducible gene expression experiments, into seeds. While the detection of a reporter gene at the early imbibitional stages could be replaced by reverse transcription-polymerase chain reaction (RT-PCR), the interference of entry of the chemical ligand into seeds by the testa was still problematic to gene induction experiments. To develop an efficient inducible expression system for gene function analysis in seeds, an inducible expression system with nitrate, which is a testa-permeable ligand, was examined. The vector containing the 2.1-kb upstream sequence of NITRITE REDUCTASE 1 was able to cause expression of a test gene (long non-coding RNA) in imbibed seeds at the stage before testa rupture in a nitrate-dependent manner. This system can be used not only for characterization of genes associated with seed dormancy and germination in basic research, but also for the development of germination recovery or enhancement technologies for agricultural applications.