Holtkotte Xu, Ponnu Jathish, Ahmad Margaret, Hoecker Ute
Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), Biocenter, University of Cologne, Cologne, Germany.
UMR 8256 (B2A) CNRA-UPMC, IBPS, Université Pierre et Marie Curie, 9 quai Saint Bernard, Paris, France.
PLoS Genet. 2017 Oct 9;13(10):e1007044. doi: 10.1371/journal.pgen.1007044. eCollection 2017 Oct.
Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that-in turn-COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.
植物不断调整其生长、发育和新陈代谢以适应周围的光照环境。拟南芥光感受器CRY1和CRY2可感知蓝光，随后通过抑制COP1/SPA E3泛素连接酶来启动光信号转导。虽然隐花色素与SPA之间的相互作用依赖蓝光，但有人提出CRY1与COP1组成性相互作用，即在黑暗中也是如此。在这里，我们的体内共免疫沉淀实验表明，只有在幼苗暴露于蓝光后，CRY1和CRY2才与COP1形成复合物。在黑暗生长的幼苗中未观察到COP1与CRY1或CRY2之间的关联。因此，我们的结果表明，隐花色素在被蓝光光激活后与COP1/SPA复合物结合。在缺乏所有四种SPA蛋白的spa四重突变体中，CRY1和COP1在体内不相互作用，无论是在黑暗生长的幼苗还是蓝光生长的幼苗中。因此，SPA蛋白是蓝光下CRY1与COP1高亲和力相互作用所必需的。酵母三杂交实验也表明SPA1增强了CRY1-COP1的相互作用。负责与COP1结合的SPA1卷曲螺旋结构域对于在体内介导CRY1-SPA1相互作用是必需的，这意味着反过来COP1可能是CRY1-SPA1复合物形成所必需的。因此，SPA1和COP1可能协同作用以识别和结合光激活的CRY1。相比之下，CRY2与COP1之间的蓝光诱导关联在体内不依赖于SPA蛋白。同样，ΔCC-SPA1与CRY2相互作用，尽管亲和力比野生型SPA1低得多。总之，我们的结果表明，CRY1和CRY2在蓝光诱导的与COP1/SPA复合物的相互作用上有很大差异。
