Fig. 3.

CsHEC1 directly activates the expression of the auxin biosynthesis gene CsYUC4 to promote fruit neck elongation. (A and B) Gene expression analysis of CsYUC4 in Cshec1 knockout mutants (A) and CsHEC1-OE lines (B). Values are means ± SD (n = 3). *P < 0.05, **P < 0.01 (Student’s t test). (C) Schematic diagram of the putative E-box cis-elements in the CsYUC4 promoter. Asterisks represent the locations of putative E-box cis-elements. Black lines represent the fragments used for ChIP-PCR in E. (D) Yeast one-hybrid analysis of the interaction between the CsHEC1 protein and the variant E box from the CsYUC4 promoter. The interaction between IND-AD and the PID-E box was used as a positive control. (E) CsHEC1 binds to the cis-element P1 regions of the CsYUC4 promoter in vivo by ChIP-PCR analysis. Tubulin was used as a negative control. (F) Firefly luciferase (LUC) and renilla reiformis luciferase (REN) activity measurement was performed in N. benthamiana leaves by coexpression of Pro35S:CsHEC1 or Pro35S:CsHEC2 and ProCsYUC4:LUC. The empty vector pGreenII 62-SK was used as the control. Values are means ± SD (n = 3 in E; n = 5 in F). **P < 0.01 (Student’s t test). (G) Electrophoretic mobility-shift experiment showed that CsHEC1 binds to the P1 fragment of the CsYUC4 promoter. (H) Expression analysis of CsYUC4 in the ProCsHEC1:CsYUC4 transgenic lines. (I and J) Fruit morphology at 10 DPP (I) and 40 DPP (J) in WT and ProCsHEC1:CsYUC4 plants. The brackets indicate the fruit neck; the double arrows represent the measured FNL. (Scale bars, 2 cm.) (K and L) Quantification of FNL (K) and ratio of FNL/FL (L) in WT and ProCsHEC1:CsYUC4 lines at 40 DPP. (M) IAA content in the fruit neck of WT and ProCsHEC1:CsYUC4 plants. Values are means ± SD (n = 3 in H and M; n = 7 in K and L). Significance analysis compared with WT was performed with the two-tailed Student’s t test (*P < 0.05, **P < 0.01).