Xie Lisi, Lang-Mladek Christina, Richter Julia, Nigam Neha, Hauser Marie-Theres
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
Plant Physiol Biochem. 2015 Aug;93:18-28. doi: 10.1016/j.plaphy.2015.03.006. Epub 2015 Mar 23.
The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV Resistance Locus 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is Constitutively Photomorphogenic 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B.
拟南芥中紫外线B诱导的ARIADNE12（ARI12）基因是E3泛素连接酶的RING-中间-RING（RBR）家族成员，在哺乳动物同源物中已鉴定出其新的泛素化机制。这种RING-HECT混合机制需要一个保守的半胱氨酸，而在ARI12中该半胱氨酸被丝氨酸取代，这可能会影响E3泛素连接酶的活性。我们已经表明，在光形态建成的紫外线B条件下，ARI12是经典紫外线B（UV-B）抗性位点8（UVR8）途径的下游靶点。然而，在高剂量率的UV-B条件下，ARI12的诱导独立于UVR8以及UV-A/蓝光和红/远红光光感受器。几种光信号通路的一个关键成分是组成型光形态建成1（COP1）。在UV-B照射下，COP1通过与UVR8相互作用被困在细胞核中，从而激活调控UV-B保护性代谢物生物合成和生长适应的基因。为了阐明COP1在调控ARI12 mRNA表达以及ARI12蛋白稳定性、定位和与COP1相互作用方面的作用，我们在有和没有UV-B的情况下评估了它们之间的关系。我们发现，COP1在白光、低剂量率和高剂量率的UV-B条件下都对ARI12有调控作用。此外，我们还表明，ARI12确实是一种E3泛素连接酶，它会发生单泛素化，这是RING-HECT混合机制的一个先决条件。最后，用表达基因组pmARI12:ARI12-GFP构建体的转基因进行的遗传分析证实了在对高剂量率UV-B的生长反应中，COP1和ARI12之间存在上位性相互作用。
