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Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

RF2a is a bZIP transcription factor that regulates expression of the promoter of rice tungro bacilliform badnavirus. RF2a is predicted to include three domains that contribute to its function. The results of transient assays with mutants of RF2a from which one or more domains were removed demonstrated that the acidic domain was essential for the activation of gene expression, although the proline-rich and glutamine-rich domains each played a role in this function. Studies using fusion proteins of different functional domains of RF2a with the 2C7 synthetic zinc finger DNA-binding domain showed that the acidic region is a relatively strong activation domain, the function of which is dependent on the context in which the domain is placed. Data from transgenic plants further supported the conclusion that the acidic domain was important for maintaining the biological function of RF2a. RF2a and TBP (TATA-binding protein) synergistically activate transcription in vitro (Zhu, Q., Ordiz, M. I., Dabi, T., Beachy, R. N., and Lamb, C. (2002) Plant Cell 14, 795-803). In vitro and in vivo assays showed that RF2a interacts with TBP through the glutamine-rich domain but not the acidic domain. Functional analysis of such interactions indicates that the acidic domain activates transcription through mechanisms other than via the direct recruitment of TBP.

TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key factors for the assembly of eukaryotic transcription initiation complexes. We used a rice whole-cell extract in vitro transcription system to characterize the functional interactions of recombinant plant TBP and TFIIB. Bacterially expressed rice TBP (OsTBP2) bound to the TATA box of the rice pal gene encoding phenylalanine ammonia-lyase, caused DNA bending, and enhanced basal transcription from the pal promoter in a TATA box-dependent manner. Recombinant rice TFIIB (OsTFIIB) stimulated the DNA binding and bending activities of OsTBP2 and synergistically enhanced OsTBP2-mediated transcription from the pal promoter and the promoter of Rice tungro bacilliform virus but not from the barley pr1 promoter. We also demonstrate a physical interaction between OsTBP2 and RF2a, a rice bZIP transcription factor that bound to the box II cis element of the promoter of Rice tungro bacilliform virus, resulting in enhanced transcription from the viral promoter. Enhancement of rice whole-cell extracts with recombinant transcription factors thus provides a powerful tool for the in vitro determination of plant gene regulation mechanisms. We conclude that OsTBP2 undergoes promoter-specific functional interactions with both the basal transcription factor OsTFIIB and the accessory transcription factor RF2a.