Hartmann U, Valentine W J, Christie J M, Hays J, Jenkins G I, Weisshaar B
Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Köln, Germany.
Plant Mol Biol. 1998 Mar;36(5):741-54. doi: 10.1023/a:1005921914384.
To identify DNA sequences of the Arabidopsis thaliana chalcone synthase gene (CHS) concerned with induction by UV-B and UV-A/blue light, AtCHS promoter constructions were assayed by transient expression in protoplasts prepared from two different lines of cultured A. thaliana cells. The protoplasts responded similarly to A. thaliana leaf tissue in light-dependent CHS transcript accumulation. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light-responsive promoter activity. A 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion 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 LRUPcCHS, (light-responsive unit of the Petroselinum crispum CHS promoter). This A. thaliana CHS promoter region, designated LRUAtCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in LRUAtCHS corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of the 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 light regulation of this promoter. Since no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant, we conclude that the two phototransduction pathways regulate transcription factors which interact with common promoter elements. The results from-our analysis of a A. thaliana light-responsive promoter will facilitate the study of light-dependent gene regulation by genetic means in Arabidopsis thaliana.
为了鉴定拟南芥查尔酮合酶基因（CHS）中与UV-B及UV-A/蓝光诱导相关的DNA序列，通过在由两种不同的拟南芥培养细胞系制备的原生质体中进行瞬时表达，对AtCHS启动子构建体进行了检测。原生质体在依赖光的CHS转录本积累方面对拟南芥叶组织的反应相似。报告酶β-葡萄糖醛酸酶（GUS）用于监测光响应启动子活性。一个1972 bp的启动子赋予了UV-B及UV-A/蓝光对GUS活性的诱导作用。缺失至164 bp导致启动子强度降低，但对UV-B及UV-A/蓝光的响应得以保留。进一步缺失则消除了转录活性。164 bp的启动子包含与LRUPcCHS（皱叶欧芹CHS启动子的光响应单元）极为相似的序列。这个拟南芥CHS启动子区域，命名为LRUAtCHS，足以赋予异源核心启动子UV-B及UV-A/蓝光响应性。LRUAtCHS中与LRUPcCHS的ACGT元件及MYB识别元件相对应的序列发生突变，导致164 bp和335 bp启动子缺失失活。然而，突变的668 bp启动子保留了UV-B及UV-A/蓝光诱导的残余表达，表明在-335上游存在其他功能序列。-442附近单个类G-box序列的突变对光响应性没有影响，表明其在该启动子的光调控中不起作用。由于未观察到任何启动子变体对UV-B及UV-A/蓝光的响应存在差异，我们得出结论，两条光转导途径调控与共同启动子元件相互作用的转录因子。我们对拟南芥光响应启动子的分析结果将有助于通过遗传学方法研究拟南芥中依赖光的基因调控。
