Figure 4.

GhTCE1 binds to the promoters of GhLTP2 and GhLTP3 to activate their expression. A and B, LUC signals detected in tobacco leaves. Promoters of GhLTP2 and GhLTP3 (LTP2_pro and LTP3_pro) were separately fused upstream of LUC in the vector pGreenII 0800-LUC and designated LPT2_pro:LUC and LPT3_pro:LUC. As an internal control, 35S_pro:REN in vector pGreenII 0800-LUC was used. GhTCE1 and ΔGhTCE1 were fused downstream of the 35S promoter in pGreenII 62-SK. The pGreenII 62-SK empty vector was used as negative control and named Null. For each tobacco leaf, half was used to express pGreenII 0800-LUC:LTP_pro+pGreenII 62-SK:GhTCE1+35S_pro:REN (named GhTCE1/LTP_pro) and half was used to transiently express pGreenII 0800-LUC:LTP_pro+pGreenII 62-SK empty vector:REN (named Null/LTP_pro) following Agrobacterium-mediated transfection, ΔGhTCE1 in the same way. C, Activated expression of LTP2_pro and LTP3_pro by GhTCE1 in cotton callus protoplasts using the LUC expression system. Relative activation level represents the ratio of fluorescence intensity of LUC/REN with GhTCE1 to LUC/REN without GhTCE1. D, Y1H assays of GhTCE1 binding to LTP2_pro and LTP3_pro. The full-length coding sequence of GhTCE1 was fused to the pGADT7 vector, and LTP2_pro and LTP3_pro were cloned into the pHis2 vector. Interaction was determined on selective medium lacking Trp+Leu+His in the presence of 3-AT. E, A series of truncations (LTP2_proΔ1 to LTP2_proΔ4) with E-box deletions were used in the LUC activation system vector. F, GhTCE1 activates the expression of truncated LTP2_pro by detected LUC signals on tobacco leaves. The truncated LTP2_pro constructs were fused to the vector pGreenII 0800-LUC, and combined with pGreenII 62-SK:GhTCE1+35S_pro:REN as a positive control. The pGreenII 62-SK empty vector was used as a negative control. They are labeled the same way as in (A) and (B). G, GhTCE1 activates the expression of truncated LTP3_pro (LTP3_proΔ1 andLTP3_proΔ2) by LUC signals detected on tobacco leaves. H, GhTCE1 activates the expression of truncated LTP3_pro (LTP3_proΔ1 and LTP3_proΔ2) by LUC signals detected on tobacco leaves. The process same to (F). I, The probe sequences of LTP2_pro and LTP3_pro are noted and the E-boxes are shown in red. J and K, EMSA showing the specificity of in vitro binding between GhTCE1 and the E-box elements of LTP2_pro (J) and LTP3_pro (K). The WT probes were subjected to biotin-labeling. No GhTCE1-His tag protein lines as control. The arrows indicate the up-shifted binds. L and M, Validation of the binding of GhTCE1 to the promoters E-box elements of LTP2_pro (L) and LTP3_pro (M) by ChIP-qPCR. Error bars in (C), (L), and (M) represent ± standard errors of three biological replicates (10 samples each in [C], three samples each in [L] and [M]). Statistical significance is indicated by *P < 0.05, **P < 0.01, Supplemental Data Set S2.