UV and blue light regulation of transcription of the chalcone synthase gene in Arabidopsis

In order to identify light responsive cis-acting DNA sequence elements of the Arabidopsis thaliana chalcone synthase gene (AtCHS) concerned with induction by UV-B and UV-A/blue light, we developed a UV/blue light inducible transient expression system. This system involved transfection of chimaeric AtCHS promoter constructs into Arabidopsis cell culture protoplasts. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light responsive promoter activity. Development of this system involved defining and optimising procedures for preparing protoplasts from the Arabidopsis cell culture, transfection of chimaeric AtCHS promoter constructs into the protoplasts and incubation under defined illumination conditions. An efficient homologous protoplasts transient expression system was developed which subsequently enabled us to undertake functional analysis of the AtCHS promoter. Application of the transient expression system allowed us to define the light responsive cis-acting elements concerned with the transcriptional activation of AtCHS. This analysis showed that a 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion of the promoter to 164 bp resulted in reduced promoter strength but retention of responsiveness to UV-B and UV-A/blue light. Further deletion abolished transcriptional activity. The 164 bp promoter contains sequences closely resembling the light-responsive unit (LRUPcCHS) of the Petroselinum crispum CHS promoter. This Arabidopsis CHS promoter region, designated LRUPcCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of 164 bp and 335 bp promoter deletions. However, the mutant 668 bp promoter retained residual UV-B and UV-A/blue light-induced expression, indicating the presence of additional functional sequences upstream of -335. Mutation of a single G-box-like sequence around -442 had no effect on light responsiveness, indicating that it does not function in the light regulation of this promoter. Furthermore, in this analysis no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant tested. This suggests that the UV-B and the UV-A/blue phototransduction pathways regulate transcription factors which interact with common promoter elements. These results are discussed. We employed several different approaches to investigate the role of cryptochromes in the regulation of AtCHS promoter activity. Gain of function transient expression analysis suggests that CRY1 is the primary photoreceptor mediating UV- A/blue light induction of promoter activity. In addition, northern analysis using various cryptochrome mutant lines, including a cry1/cry2 double mutant, supports this hypothesis and, furthermore, suggests that (an)other photoreceptor(s) able to respond to UV-A/blue light, exist in Arabidopsis. Possible reasons for these observations are discussed. We used the transient expression system to investigate the role of sugar regulation in the control of AtCHS promoter activity in Arabidopsis protoplasts. This revealed that sucrose, glucose or fructose was required to stimulate AtCHS promoter activity. Furthermore, studies using glucose analogues were performed which suggest that the initial signal for sugar stimulation of AtCHS promoter activity is consistent with signalling by hexokinase phosphorylation of hexose sugars. Evidence for this hypothesis is discussed. (Abstract shortened by ProQuest.).