AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening
Fig 10
Dimerization of the ARF2A protein and its interaction.
ARF2A was cloned downstream of the DNA-binding domain (DB-ARF2A) and co-transformed into yeast with either (A) ARF2A cloned downstream of the activation domain (AD-ARF2A); or (B) ASR1 cloned downstream of the activation domain (AD-ASR1), yeast growth on media lacking leucine, tryptophan, histidine and adenine indicated positive protein-protein interactions. (C) Relative expression levels of ASR1 in WT cv. MicroTom fruit at five developmental stages (IG: immature green; MG: mature green; Br: breaker; Or: orange; and R: red), error bars represent SE; statistical significance was evaluated using an ANOVA test (JMP software, SAS) with three biological repeats based on the average of three technical replicates, values indicated by the same letter (a,b,c) are not statistically significant, p-value<0.05. (D) A Bimolecular Fluorescence Complementation assay (BiFC) was carried out by transient expression in tobacco leaves; ARF2A was cloned downstream of the amino-terminal region of YFP (yellow fluorescent protein; YN-ARF2A) and ASR1 was cloned downstream of the carboxy-terminal region of YFP (YC-ASR1); leaf regions were examined for fluorescent signal by light and confocal fluorescence microscopy. Inset zoom region shows that the ARF2A-ASR1 interaction is nuclear localized. Scale bars in the light and confocal fluorescence microscopy represent 50 μm and 10 μm, respectively.