Zhang Hongliang, Janina Nadežda, Ütkür Koray, Manivannan Thirishika, Zhang Lei, Wang Lizhen, Grefen Christopher, Schaffrath Raffael, Krämer Ute
Molecular Genetics and Physiology of Plants, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum 44801, Germany.
Microbiology, Institute for Biology, University of Kassel, Kassel 34132, Germany.
Plant Physiol. 2025 Mar 28;197(4). doi: 10.1093/plphys/kiaf128.
Diphthamide is a posttranslationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR 2 (eEF2) and the target of diphtheria toxin in human cells. In yeast and mammals, the 4Fe-4S cluster-containing proteins Dph1 and Dph2 catalyze the first biosynthetic step of diphthamide formation. Here, we identify Arabidopsis (Arabidopsis thaliana) DPH2 and show that it is required for diphthamide biosynthesis, localizes to the cytosol, and interacts physically with AtDPH1. Arabidopsis dph2 mutants form shorter primary roots and smaller rosettes than the wild type, similar to dph1 mutants which we characterized previously. Additionally, increased ribosomal -1 frameshifting error rates and attenuated TARGET OF RAPAMYCIN (TOR) kinase activity in dph2 mutants also phenocopy the dph1 mutant. Beyond the known heavy metal hypersensitivity and heat shock tolerance of dph1, we show here that both dph1 and dph2 mutants are hypersensitive to elevated light intensities and oxidative stress and that wild-type Arabidopsis seedlings accumulate diphthamide-unmodified eEF2 under oxidative stress. Both mutants share the deregulation of 1,186 transcripts associated with several environmental and hormone responses. AtDPH1 and AtDPH2 do not complement the corresponding mutants of Saccharomyces cerevisiae. In summary, DPH2 and DPH1 interact to function inter-dependently in diphthamide formation, the maintenance of translational fidelity, wild-type growth rates, and TOR kinase activation, and they contribute to mitigating damage from elevated light intensities and oxidative stress. Under oxidative stress, a dose-dependent loss of diphthamide could potentiate downstream effects in a feed-forward loop. This work advances our understanding of translation and its interactions with growth regulation and stress responses in plants.
白喉酰胺是真核生物翻译延伸因子2（eEF2）翻译后修饰的组氨酸残基，也是人类细胞中白喉毒素的作用靶点。在酵母和哺乳动物中，含4Fe-4S簇的蛋白质Dph1和Dph2催化白喉酰胺形成的第一步生物合成反应。在此，我们鉴定了拟南芥（Arabidopsis thaliana）中的DPH2，并表明它是白喉酰胺生物合成所必需的，定位于细胞质，且与AtDPH1存在物理相互作用。与我们之前鉴定的dph1突变体相似，拟南芥dph2突变体形成的初生根比野生型短，莲座叶比野生型小。此外，dph2突变体中核糖体-1移码错误率增加以及雷帕霉素靶蛋白（TOR）激酶活性减弱，也与dph1突变体表型相似。除了已知的dph1对重金属过敏和耐热激外，我们在此表明dph1和dph2突变体均对高光强度和氧化应激敏感，且野生型拟南芥幼苗在氧化应激下会积累未修饰白喉酰胺的eEF2。这两种突变体共有1186个与多种环境和激素反应相关的转录本失调。AtDPH1和AtDPH2不能互补酿酒酵母的相应突变体。总之，DPH2和DPH1相互作用，在白喉酰胺形成、维持翻译保真度、野生型生长速率和TOR激酶激活中相互依赖发挥作用，并且它们有助于减轻高光强度和氧化应激造成的损害。在氧化应激下，白喉酰胺的剂量依赖性损失可能会在前馈环中增强下游效应。这项工作增进了我们对植物翻译及其与生长调节和应激反应相互作用的理解。
