Fig. 8.

Cross-regulation of endogenous StBEL6, -34, and -22 in GAS:BEL5 overexpression lines. The movement of transgenic StBEL5 mRNA from leaf to primary and secondary roots was confirmed previously using transgenic lines expressing full-length StBEL5 RNA driven by the GAS promoter of melon (Cucumis melo) grown under SD conditions (Fig. 1B; Lin et al., 2013). This promoter is expressed in the minor veins of leaf mesophyll (Ayre et al., 2003; Banerjee et al., 2009). Substantial amounts of transgenic BEL5 RNA moved into primary and secondary roots and activated accumulation of WT StBEL5 transcripts (Fig. 7; Lin et al., 2013). This same RNA was used to assess levels of endogenous RNA for StBEL6, -34, -22, and -14 in both the WT (open bars) and transgenic BEL5 line (grey shaded bars) in leaves, primary (1° Root) and secondary (2° Root) roots. The existing upstream double TGAC core motifs are shown for each gene. The BEL14 upstream sequence contains no tandem TGAC motif and was included as a negative control. One-step RT-PCR was performed using 200–250ng of total RNA and gene-specific primers for StBEL6, -34, -22, and -14. All PCRs were standardized and optimized to yield product in the linear range. Homogenous PCR products were quantified using ImageJ software (Abramoff et al., 2004) and normalized using 18S rRNA values. Standard errors of the means of three replicate samples are shown. The asterisk indicates a significant difference (P<0.05) using Student’s t-test.