Li Qiaomu, Wang Le, Grubb Lauren E, Talasila Mohana, Rodriguez Gallo Maria Camila, Mehta Devang, Scandola Sabine, Uhrig Richard Glen
Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E9, Canada.
Department of Biosystems, Katholieke Universiteit Leuven, B-3001, Leuven, Belgium.
New Phytol. 2025 Jul;247(2):897-915. doi: 10.1111/nph.70192. Epub 2025 May 20.
The timing of flowering is a critical agronomic trait governed by an extensive and sophisticated regulatory network. To date, limited understanding of how posttranslational modifications regulate flowering time exists. Here, using Arabidopsis, we resolve a role for the B4 Raf-like MAPKKK protein kinase RAF24 in regulating flowering time. Loss of RAPIDLY ACCELERATED FIBROSARCOMA24 (RAF24) led to premature flowering time through altered expression of FLC and FT. Comparative phosphoproteomic analysis of raf24 and wild-type plants revealed a list of known flowering-related phosphoproteins from distinct flowering pathways displaying downregulated phosphorylation. Of these, the RING-type ubiquitin ligase HISTONE MONO-UBIQUITINATION 2 (HUB2) lacked phosphorylation in the absence of RAF24. Absence of RAF24 induced H2Bub1 overaccumulation, with protein-protein interactome analysis of HUB2 in the presence and absence of RAF24 influencing HUB2 protein interaction partners, such as H2B. HUB2 was also found to physically interact with SUCROSE NONFERMENTING KINASE 2.4 (SnRK2.4) and SnRK2.6, known substrates of RAF24. Using phospho-mimetic and phospho-ablative plant lines, we then validated the importance of HUB2 phosphorylation at serine 314 (S314) in maintaining appropriate flowering time. Our findings uncovered a novel biological role of RAF24, as a higher-order flowering regulator, while further implicating HUB2 as a centerpiece of flowering time regulation.
开花时间是一个关键的农艺性状，受广泛而复杂的调控网络控制。迄今为止，对于翻译后修饰如何调控开花时间的了解有限。在此，我们以拟南芥为研究对象，解析了B4 Raf样丝裂原活化蛋白激酶激酶激酶（MAPKKK）蛋白激酶RAF24在调控开花时间中的作用。快速加速纤维肉瘤24（RAF24）缺失导致开花时间提前，这是通过改变开花位点C（FLC）和开花素（FT）的表达实现的。对raf24和野生型植物进行比较磷酸化蛋白质组分析，揭示了一系列来自不同开花途径的已知开花相关磷酸化蛋白，其磷酸化水平下调。其中，在缺乏RAF24时，环型泛素连接酶组蛋白单泛素化2（HUB2）缺乏磷酸化。RAF24缺失诱导H2B单泛素化（H2Bub1）过度积累，对存在和不存在RAF24时的HUB2进行蛋白质-蛋白质相互作用组分析，发现其影响HUB2的蛋白质相互作用伙伴，如H2B。还发现HUB2与蔗糖非发酵激酶2.4（SnRK2.4）和SnRK2.6存在物理相互作用，而这两种蛋白是RAF24的已知底物。然后，我们使用磷酸模拟和磷酸去除植物株系，验证了HUB2丝氨酸314（S314）位点磷酸化在维持适当开花时间方面的重要性。我们的研究结果揭示了RAF24作为一种高阶开花调节因子的新生物学作用，同时进一步表明HUB2是开花时间调控的核心组成部分。
