UV/blue light signal transduction regulating gene expression in Phaseolus vulgaris and Arabidopsis thaliana

Abstract

Plants regulate the expression of some genes in response to ultraviolet (UV) and blue light. To investigate the signal transduction pathways regulating UV/blue light regulated gene expression, the effects of specific inhibitors were examined in protoplasts isolated from dark-adapted P. vulgaris leaves and a photomixotrophic Arabidopsis cell culture. Pharmacological studies with P. vulgaris protoplasts indicate that calcium, protein phosphorylation and dephosphorylation and protein synthesis are required for UV-A/blue light regulation of genes encoding the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase (rbcS), which is similar to the UV-A/blue and UV-B light signalling components regulating chalcone synthase (CHS) expression in Arabidopsis cells. Further studies were carried out in Arabidopsis cells to characterise the UV-A/blue and UV-B regulation of CHS. A 5 min UV-B illumination, followed by transfer to non-inductive, low fluence rate white light for 6 hours, was sufficient to induce CHS transcripts. In contrast, a one hour UV- A/blue illumination, followed by transfer to non-inductive light for 5 hours was required before any CHS transcript accumulation was detected. This indicates that the two pathways are distinct. However, both the UV-A/blue and UV-B light regulation of gene expression appears to involve plasma membrane redox activity, because the impermeable electron acceptor ferricyanide (FeCN), strongly inhibited UV-A/blue and UV-B phototransduction. Additionally, the flavoprotein inhibitor diphenylene iodonium (DPI), strongly inhibited UV-A/blue and UV-B induced CHS and PAL expression. These results suggest that the phototransduction pathways require at least one flavoprotein-mediated electron transfer step as a signalling component. Expression of the Arabidopsis gene encoding the calmodulin-like protein TOUCH3 (TCH3) is induced by FeCN and DPI. Cells treated with a calcium ionophore were not altered in UV/blue light regulated CHS expression. This indicated that cytosolic calcium increases induced by FeCN and DPI are not inhibiting UV/blue phototransduction. However, UV-A/blue and UV-B light inhibited the ionophore-induced expression of TCH3 in Arabidopsis cells. This implied that UV-A/blue and UV-B light were activating a calcium efflux mechanism, lowering cytosolic calcium concentrations. Therefore, the effect of an inhibitor of calcium-ATPases, erythrosin B (BB), was examined. EB prevented the UV-A/blue light induction of CHS. However, EB had no effect on the UV-B induction of CHS. Further pharmacological studies were carried out to characterise the UV/blue inhibition of TCH3 expression in the presence of ionophore. Transgenic Arabidopsis wild-type and hy4 mutant plants expressing the cytosolic calcium reporter protein, aequorin, were generated. UV-A/blue light induced an increase in aequorin luminescence in both transgenic lines, indicating that UV-A/blue light induces an increase in cytosolic calcium concentration, but this response is not mediated by CRY1.No effect on the regulation of CHS expression by reactive oxygen species (ROS) and scavengers of ROS was observed, suggesting that the production of ROS by plasma membrane redox processes is not a component of the signalling pathways. Interestingly, UV-A/blue and UV-B light strongly induced GST5 transcripts in the cell culture and plants. The experiments described in this thesis are discussed and a hypothesis for the signal transduction processes involved in UV/blue regulated gene expression is presented