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Fig. 3. LFY binds to sequences within the distal early element of the AP3 promoter. (A) Electrophoretic mobility shift assay demonstrates that in vitro translated LFY gene product binds to a labeled sequence corresponding to the AP3 promoter region from –727 to –554 (test fragment) which contains the DEE (shown in gray). The ability of various cold competitors to block LFY binding is shown. Competitor 1 contains sequences from –727 to –554 and corresponds to the test fragment; competitor 2, –705 to –587; competitor 3, –662 to –554; competitor 4 –727 to –626; competitor 5, –618 to –554; and competitor 6, –727 to –678. The brome mosaic virus (BMV) in vitro translation reaction was used as a non-specific control. (B) The region to which LFY binds was further subdivided into site I (–678 to –659) and site II (–658 to –627). Palindromic sequences within each of these sites are underlined; these sites are aligned with LFY binding sites defined in the AP1 and AG floral homeotic gene regulatory regions (Busch et al., 1999 ; Parcy et al., 1998). Sequences mutated in AP3 site I and AP3 site II are shown in bold. (C) Yeast one-hybrid assays demonstrate that LFY binds to AP3 site I. Trimerized versions of AP3 site I, site II or the 52 bp fragment containing both sites (site I site II) were fused in a normal (+) or inverted (–) orientation to the lacZ reporter gene and introduced into yeast. Similar constructs were generated containing the mutated site Im or site IIm versions described in B. The ability of the GAL4 activation domain alone (GAD) or the GAL4 activation domain fused to LFY (LFYAD) to activate lacZ expression was assayed in five replicates. Standard errors for each construct are shown. (D) Electrophoretic mobility shift assays demonstrate that LFY binds to site I. The ability of in vitro translated LFY protein to bind to labeled sequences corresponding to site I, site II, or the mutated versions was assessed. BMV, non-specific control.
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