A WRKY Transcription Factor Recruits the SYG1-Like Protein SHB1 to Activate Gene Expression and Seed Cavity Enlargement
Figure 8
SHB1 activates transcription from MINI3 and IKU2 promoters.
(A) A diagram showing the effector, reporter, and reference constructs used in the transient trans-activation assays. Full-length MINI3 fused to CFP was used as effector and was driven by the CaMV 35S promoter, and an empty vector (EV) was used as a control. For reporters, the GUS gene was driven by either the wild-type MINI3 or IKU2 promoter or mutated W1-box in the MINI3 promoter or mutated Wi-box in the IKU2 promoter and used as a reporter. Arrowheads indicate transcription start sites, and NOS-T represents polyadenylation signal from the nopaline synthase gene. The asterisk indicates the location of W-boxes in the MINI3 or IKU2 promoters. LUC gene driven by the CaMV 35S promoter was used as an internal reference. (B) GUS expression from the MINI3 promoter with a wild-type (pMINI3::GUS) or a mutated (pMINI3m1::GUS) W1-box in the presence of an empty vector (EV) or MINI3 in mini3/shb1, mini3/SHB1 or mini3/shb1-D backgrounds. (C) GUS expression from the IKU2 promoter with a wild-type (pIKU2::GUS) or a mutated (pIKU2mwi::GUS) Wi-box in the presence of an EV or MINI3 in mini3/shb1, mini3/SHB1 or mini3/shb1-D bckgrounds. GUS activities were expressed in pico-moles per min per mg protein relative to LUC activities, which were expressed in light units per mg protein. The relative GUS/LUC activities for the EV and pMINI3::GUS pair (24 pico-moles/min) in B or the EV and pIKU2::GUS pair (52 pico-moles/min) in C were set to 1, and the remaining values were expressed as fold trans-activation. Data are calculated from at least three independent experiments as the mean plus or minus standard error (n≥3). The levels of significance of differences were determined using Student's t-test. ** P<0.01, * P<0.05, and ns, not significantly different.