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. 2005 Dec;17(12):3470–3488. doi: 10.1105/tpc.105.035659

Figure 2.

Figure 2.

The N-Terminal Conserved Region of AREB1 Functions as a Transcriptional Activation Domain in Protoplasts Derived from Arabidopsis T87 Cultured Cells.

All transactivation experiments were performed 3 to 10 times, and results from one representative experiment are shown. Bars indicate standard deviation; n = 3 to 5.

(A) Scheme of the effector and reporter constructs used in the transactivation analysis with AREB1 bZIP DNA binding domain. The effector constructs contain the CaMV 35S promoter and TMV Ω sequence fused to AREB1 cDNA fragments encoding different portions of AREB1. The reporter construct, RD29B-GUS, contains 77-bp fragments of the RD29B promoter connected tandemly five times. The promoters were fused to the −51 RD29B minimal TATA promoter–GUS construct. Nos-T, nopaline synthase terminator.

(B) Transactivation domain analysis of AREB1 using N-terminal deletion constructs. Protoplasts were cotransfected with the RD29B-GUS reporter and the effector construct (shown on the left) carrying an N-terminal truncated form of AREB1 cDNA or pBI-35SΩ (vector). To normalize for transfection efficiency, the pBI35SΩ-LUC reporter was cotransfected as a control in each experiment. Bars indicate standard deviation of three replicates. “Relative activity” indicates the multiples of expression compared with the value obtained with the pBI221-35SΩ vector control. Top numbers indicate amino acid numbers of AREB1. P, Q, R, S, T, and U indicate the partial region of the AREB1 cDNA. The region P, Q, R, or U includes a conserved domain (solid rectangle), C1, C2, C3, or C4, respectively, in Figure 1A.

(C) Schematic diagram of the effector and reporter constructs used in the transactivation analysis with the GAL4 DNA binding domain. The effector plasmids encoding the GAL4 DNA binding domain are fused to AREB1 cDNA fragments encoding different portions of AREB1. The GUS reporter construct, GAL4-GUS, containing GAL4 binding sites, is fused to the minimal promoter of CaMV 35S.

(D) N-terminal conserved P region of AREB1 contains sufficient domain for transcriptional activation. Protoplasts were cotransfected with the GAL4-GUS reporter and the effector construct expressing a portion of AREB1 or the vector DNA.

(E) AREB1ΔQT is a constitutive active form of AREB1. Protoplasts were cotransfected with the RD29B-GUS reporter and the effector construct expressing intact AREB1, AREB1ΔQT, or AREB1ΔP/RT, or vector DNA.