Journal: Horticulture Research
Article Title: Involvement of the LcARF17- and LcRAP2-4-LcLOX7 regulatory modules in the biosynthesis of fresh aroma in litchi aril
doi: 10.1093/hr/uhag010
Figure Lengend Snippet: LcARF17 and LcRAP2-4 directly activate LcLOX7 transcription. (A) Subcellular localization of LcARF17 and LcRAP2-4 in tobacco leaf epidermal cells. The nuclear localization marker (At1g22590) was detected via mCherry fluorescence, GFP signals were visualized via GFP fluorescence, and a bright-field image was also captured. Merged images show the overlays of mCherry, GFP, and bright-field signals. Scale bars = 20 μm. (B) Transcriptional activation assay of LcARF17 and LcRAP2-4 in tobacco leaves. Bar chart quantifying the inherent transcriptional activation activity of LcARF17 and LcRAP2-4. pBD-Empty and pBD-VP16 were used as negative and positive control, respectively. (C) Transient dual-luciferase assays showing activation of LcLOX7 promoter by LcARF17 and LcRAP2-4. Bar chart quantifying the enhanced activity of the LcLOX7 promoter when co-expressed with LcARF17 and LcRAP2-4. pGreen 62-SK was used as the negative control. Data are presented as mean ± SD. Statistical significance determined by Student’s t -test: * P < 0.05, ** P < 0.01. (D) In vitro binding of LcARF17 and LcRAP2-4 to the LcLOX7 promoter was demonstrated by electrophoretic mobility shift assay (EMSA). Arrows highlight shifted bands indicating DNA-protein complex and free probe formations. ‘−’ and ‘+’ represent absence or presence of the respective component; ‘++’ indicates the increasing amounts of unlabeled cold probes (for competition) or mutant probes. MBP-tagged protein alone served as a negative control. (E) Yeast one-hybrid analysis of LcARF17 and LcRAP2-4 binding to the promoter of LcLOX7 . No basal activities of LcLOX7 promoter were detected in yeast grown on SD/-Ura medium with 300 ng/ml aureobasidin A (AbA). The interaction was evaluated based on the growth conditions of transformed yeast on SD/-Leu medium containing 300 ng/ml AbA.
Article Snippet: Subsequently, the yeast one-hybrid assay was conducted using the MatchmakerTM Gold Yeast One-Hybrid Library Screening System (BD Clontech).
Techniques: Marker, Fluorescence, Activation Assay, Activity Assay, Positive Control, Luciferase, Negative Control, In Vitro, Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Transformation Assay
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