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