Heilmann Monika, Velanis Christos N, Cloix Catherine, Smith Brian O, Christie John M, Jenkins Gareth I
Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bower Building, Glasgow, G12 8QQ, UK.
Plant J. 2016 Oct;88(1):71-81. doi: 10.1111/tpj.13260. Epub 2016 Sep 9.
UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet-B (UV-B) light that initiates photomorphogenic responses in plants. UV-B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt-bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt-bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt-bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV-B responses in vivo with a similar dose-response relationship to wild-type. The UV-B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV-B photoreception, initiating signal transduction and responses in plants.
紫外线抗性位点8（UVR8）是一种紫外线B（UV-B）光的光感受器，可引发植物的光形态建成反应。UV-B光感受导致二聚体UVR8迅速解离为单体，这些单体与组成型光形态建成1（COP1）相互作用以启动信号转导。对纯化的UVR8进行的实验表明，二聚体通过二聚体界面上特定带电荷氨基酸之间的盐桥相互作用得以维持。然而，关于这些带电荷氨基酸在确定植物中二聚体/单体状态和UVR8功能方面的重要性，人们了解甚少。在这里，我们评估了使用不同方法来检测UVR8的二聚体/单体状态，并表明几个盐桥氨基酸的突变会影响二聚体/单体状态、与COP1的相互作用以及UVR8在体内的光感受器功能。特别是，精氨酸286与天冬氨酸96和107之间形成的盐桥是二聚体形成的关键。将精氨酸286突变为丙氨酸会损害二聚体形成、与COP1的相互作用以及体内功能，而突变为赖氨酸则会产生一种在体内具有功能的较弱二聚体，这表明精氨酸/赖氨酸残基的正电荷对二聚体形成很重要。值得注意的是，天冬氨酸96和107保守地突变为天冬酰胺的UVR8突变体在二聚体形成方面受到严重损害，但在体内介导UV-B反应，其剂量反应关系与野生型相似。该突变体的UV-B反应性与二聚体形成和单体化无关，表明单体UVR8具有进行UV-B光感受、启动信号转导和植物反应的潜力。
