RFL is a rice ortholog of floral meristem identity genes, FLO of Anthirrhinum and LFY of Arabidopsis (Kyozuka et al. 1998). In Arabidopsis, it was shown that LFY is a major determinant of floral fate and a direct transcriptional activator of floral organ identity genes, such as AP1, AP3 and AG (Parcy et al. 1998).

Previously we reported that RFL mRNA expression was rapidly up-regulated in the meristem upon transition to reproductive development (Kyozuka et al. 1998). To understand molecular basis of this up-regulation, promoter analysis of the RFL gene was performed (Fig. 1A). A series of constructs carrying a GUS reporter gene were generated and introduced to rice plants via Agrobacterium mediated transformation, and GUS activity was monitored by histo-chemical analysis.

A reporter gene with the longest promoter, 5.6kRFLp::GUS conferred rapid and meristemspecific induction of GUS activity upon transition to the reproductive phase (Fig. 1B), indicating that the 5.6 kb upstream region from the translation initiation site contains sufficient information for the RFL expression. Deletion of the promoter to -2 kb and -600 bp did not change the pattern of GUS expression. On the other hand, none of the transgenic lines carrying 200RFLp::GUS showed detectable GUS activity, indicating that the region between -200 bp and -600 bp is required for the up-regulation (Fig. 1). To determine an enhancer region more precisely, five more constructs containing -530, -480, -420, -350, -250 bp RFL promoter region were introduced into rice plants. All the 4 lines of 420RFLp::GUS lines showed the up-regulation of GUS activity in the reproductive meristem (Fig. 1D). In contrast, four among five lines of 350RFLp::GUS analyzed showed no sign of GUS expression and one line showed

sporadic and weak GUS activity (Fig. 1F). None of the lines containing 250RFLp::GUS or 200pRFLp::GUS showed detectable GUS activity. Taken all these together, it is very likely that a 70 bp region between -350 and -420bp contains most information necessary for the meristem-specific up-regulation of RFL.

Next, we asked whether this 70 bp sequence is sufficient for the up-regulation. For this purpose, a four tandem repeat of the 70 bp region was fused with a minimal CaMV35S promoter containing TATA sequence (Benfey et al. 1990). The resultant 4x70TATA::GUS gene was transformed to rice (Fig. 2A). Five independent lines of the 4x70TATA::GUS plants were randomly chosen and used for the further analysis. A chimeric gene, designated as TATA::GUS, containing the CaMV35S minimal promoter and a GUS gene was also generated and introduced to rice as a negative control.

The 4x70TATA::GUS and TATA::GUS plants did not show any detectable GUS activity in vegetative meristems (Fig. 2B, E). After transition to the reproductive phase, three out of five 4x70TATA::GUS lines exhibited GUS expression in the meristem while none of the three TATA::GUS lines showed significant level of GUS expression throughout the development (Fig. 2C, D, F, G). Expression of the 4x70TATA::GUS genes was spatially restricted to the meristem, indicating that this 70 bp sequence contains the enhancer(s) responsive for reproductive meristem specific up-regulation of RFL.

Conclusion We identified a 70 bp region spanning -420 to -350 of RFL promoter as an enhancer which is both necessary and sufficient to confer the reproductive meristem specific up-regulation of RFL expression. Very high level of GUS expression driven by the synthetic 4x70TATA promoter suggested a possibility that this promoter may be useful for improving

reproductive phenotypes of not only rice but also other crop species.

References

Kyozuka, J., S. Konishi, K. Nemoto, T. Izawa and K. Shimamoto, 1998. Down regulation of RFL the FLO/LFY homolog of rice accompanied with panicle and branch initiation. Proc. Acad. Natl. Sci. USA 95: 1979-1982.

Parcy, F., O. Nilsson, M.A. Busch, I. Lee and D. Weigel, 1998. A genetic framework for floral patterning. Nature 395: 561-566.

Benfey, P.N., L. Ren and N.-H. Chua, 1990. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 9: 1677-1684.