FIGURE 8.

Concerted function of GBF1, HY5, and HYH in light-regulated gene expression and photomorphogenic growth. A, representative picture of dark-grown seedlings. Shown from the left are segregated wild type (WT), gbf1, hy5, hyh, hy5 hyh, gbf1 hy5, gbf1 hyh, and gbf1 hy5 hyh. B, representative picture of 6-day-old BL (30 μmol/m²/s)-grown seedlings. Shown from the left are segregated wild type (WT), gbf1, hy5, hyh, hy5 hyh, gbf1 hy5, gbf1 hyh, and gbf1 hy5 hyh. C, quantification of hypocotyl length of 6-day-old BL-grown seedlings. Approximately 30 seedlings were used for the measurement of hypocotyl length. The error bars indicate S.D. D (top), schematic representation of promoter-reporter, effectors, and internal control construct used in transient expression analysis in Arabidopsis protoplasts. The 355-bp RBCS-1A promoter upstream from the start codon was fused to the NAN reporter. Effector constructs are under the control of the CaMV 35S promoter. A Pro35S:GUS construct was used as an internal control to normalize the differences in the transfection efficiencies. D (bottom), quantification of the RBCS-1A promoter-reporter activity in the presence of given combinations of effectors in transiently transformed Arabidopsis leaf protoplasts. After transfection, protoplast samples were incubated in continuous BL (40 μmol/m²/s) for 12–14 h. Half of the protoplast samples were harvested for total RNA extraction, and the other half were used for the reporter activity measurements. The NAN/GUS activity was then normalized against the relative transcript of the effectors in each combination of the effectors. The x axis values are expressed as NAN activity relative to GUS. Given are mean and S.D. values of three replicates. n ≥ 3 independent experiments with similar results. E, a model of interaction of GBF1 with HY5 and HYH. GBF1, HY5, and HYH homodimers bind to the G-box light-responsive element. GBF1 and HYH heterodimerize with HY5 and bind to the G-box. Physical interaction of GBF1 with HYH does not lead to their heterodimerization at the G-box; however, the GBF1-HYH pool results in the reduction of GBF1 and HYH pools available for the formation of GBF1-HY5 and HY5-HYH heterodimers, respectively.