GL2 bound to the PLDα1 promoter. (a) Diagram of PLDα1 promoter region. The promoter was divided into five fragments. The fourth fragment was further divided into eight small fragments. Fragments in red indicate binding by GL2. (b) GL2 bound to PLDα1-4 region. Different regions of PLDα1 promoter were cloned into pAbAi vector and these plasmids were co-transfected into yeast Y1HGold cells with pAD-GL2. Growth of transfected yeast cells on AbA medium indicates binding of GL2 to the corresponding elements. (c) Gel shift assay to test GL2 binding of 4–1 and 4–5 segments in PLDα1 promoter. Proteins were incubated with labeled probes in the presence (+) or absence (-) of 200-fold molar excess of unlabelled competitors. A NAC binding sequence was added as a negative control. Arrow indicates protein/DNA complex. (d) GL2 binding of 4–1 and 4–5 segments in PLDα1 promoter in the presence of labeled probes plus 500-fold, 200-fold and 0-fold molar excess of unlabelled competitors (from left to right lane respectively). (e) GL2 binding to PLDα1-4 by Chromatin immunoprecipitation (ChIP) assay. ChIP was performed with try cpc (root in lane 4 and leaf in lane 5) and gl2-2 plants (lane 3) using anti-GL2 antibody. Primer sets specific for the region of PLDα1-4 were used in PCR reactions. ACTIN2 was amplified as a control. Sonicated chromatin with incubation of second antibody (anti-mouse IgG) was used as a mock control (lane 6). The supernatant of sonicated chromatin from gl2-2 and try cpc were used as input control (lane 1 and lane 2).