Figure 5.

LBD induces the removal of bHLH041 to alleviate bHLH041-mediated repression of PLT1, PLT2, and WOX5 transcription. A, B) Over-accumulation of LBD16 or LBD29 induces the disappearance of bHLH041. The fluorescent signals of bHLH041-GFP were visualized in N. benthamiana leaves transiently coexpressing bHLH041-GFP with an empty vector (EV), LBD16-FLAG (LBD16), LBD29-FLAG (LBD29), or WRKY23-FLAG (WRKY23) for 4 d (n = 10 leaves) A) and in transgenic Arabidopsis ProbHLH041:bHLH041-GFP seedlings harboring a 35S:LBD16 construct with a strong (S), intermediate (I), or weak (W) autonomous callus-forming phenotype B). Scale bars, 50 µm. C) Callus-forming and shoot-regenerating phenotypes of WT, lbd16-2, bhlh041-1, and lbd16-2 bhlh041-1, explants and derived calli (n = 20 explants). Scale bar, 10 mm. D) ChIP-qPCR assay of bHLH041 binding activity to the PLT1, PLT2, and WOX5 promoters. Ten-day-old WT and bhlh041-1 ProbHLH041:bHLH041-GFP seedlings incubated in liquid CIM for 0 and 48 h were assayed by ChIP-qPCR with anti-GFP antibody, and enrichments of the amplified F-1 to F-6 from PLT1, PLT2, or WOX5 promoters are indicated. A fragment of the UBQ10 promoter was used as a negative control. Data are shown as means ± Sd (n = 3 biological replicates). E) bHLH041 inhibits PLT1, PLT2, and WOX5 transcription. Left, diagrams of effector and reporter constructs used. Ω, translational enhancer. Arabidopsis protoplasts were cotransfected with the LUC reporter PLT1:LUC, PLT2:LUC, or WOX5:LUC with an empty vector (EV) or 35S:Ω:bHLH041-GFP construct (bHLH041) and incubated in CIM for 16 h. LUC and REN activities were determined and shown as means of original LUC/REN ratios × 100 ± Sd (n = 3 biological replicates). *P < 0.05, **P < 0.01, and ***P < 0.001 by t-test.