Diphthamide formation in Arabidopsis requires DPH1-interacting DPH2 for light and oxidative stress resistance

Diphthamide is a post-translationally 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.