Figure 3.

BLH6a is a transcriptional repressor of CAld5H2 in P. alba × P. glandulosa. (A) Effector-reporter-based gene activation/repression assay. LUC gene was driven by the P. alba × P. glandulosa CAld5H2 promoter in the reporter construct. Each TF gene from P. alba × P. glandulosa was driven by the 35S promoter in the effector construct. Effect construct and reporter construct were co-infiltrated in the tobacco leaves by Agrobacterium for the LUC activity determination. The reporter construct alone was the control. (B) Transactivation/repressor activity detections in yeast strain Y2HGold. SD/-T, single dropout medium lacking tryptophan; SD/-AHT, triple dropout medium lacking adenine, histidine, and tryptophan; BD, GAL4-binding domain. (C) Chromatin immunoprecipitation (ChIP) assays in xylem (left) and leaf (right) protoplasts. ChIP was conducted in protoplasts overexpressing FLAG and FLAG:BLH6a, respectively, and followed by qPCR. Input-FLAG and Input-BLH6a were IP using one-fiftieth of the supernatants before adding antibodies in the FLAG and FLAG:BLH6a overexpression protoplasts, respectively. IP-FLAG and IP-BLH6a were IP with FLAG antibodies in FLAG and FLAG:BLH6a overexpression protoplasts, respectively. Error bars represent SD (n = 3). **p < 0.01, determined by Student's t test.