Andor ixon charge coupled device imaging apparatus

Generation of the VviFLS4 promoter-driven luciferase (LUC) construct, agrobacterium-mediated transient expression of the promoter::LUC construct in tobacco leaves and shading treatment of the infiltrated plants were conducted according to Sun et al. [86 (link)]. The primers used for VviFLS4 promoter::LUC construction are listed in Additional file 15: Table S9. Fluorescence signals were recorded with an Andor iXon charge-coupled device (CCD) imaging apparatus (Andor Technology, Belfast, UK). The transcriptional activity of the LUC gene was detected by qPCR using previously described conditions [86 (link)]. Two housekeeping genes in tobacco, NbUbi3 and NbEF-1α, were selected as endogenous controls [98 (link), 99 (link)]. The primers used for qPCR analysis are listed in Additional file 15: Table S9. Three biological replicates and three technical replicates were run per sample. For each group of assays, at least nine leaves from three individual plants were used for CCD imaging and qPCR analysis. Significance of differences in LUC gene expression between the control and treatment groups was determined by an independent sample t-test using SPSS 20.0 for windows (SPSS Inc., Chicago, IL, USA).

The VviGT14 promoter activity was assayed in tobacco leaves. The 1287-bp promoter of VviGT14 was fused with the luciferase (LUC) reporter gene (proVviGT14::LUC), and then sub-cloned into the reconstructed binary plasmid pCAMBIA1300-LUC using the InFusion HD Cloning Kit (Clontech, CA, USA). pCAMBIA1300-LUC without any promoter (empty LUC) was used as the negative control. These constructs were confirmed by sequencing, and subsequently transformed into Agrobacterium tumefaciens strain GV3101. Then the agrobacterium suspensions infiltrations were carried out using young but fully expanded leaves of 7-week-old Nicotiana benthamiana plants. Equal amount of each agrobacterium suspensions was infiltrated into either side of the 7-week-old N. benthamiana leaf as described in our previous study [36 (link)]. Subsequently, the infiltrated plants were shifted to an artificial climatic box in dark for 12 h, and then cultured under 16 h light/8 h dark for 60 h at 25 °C. At least six biological replicates were performed. The LUC activity was determined using the Andor iXon charge-coupled device (CCD) imaging apparatus (Andor Technology, Belfast, UK).