AtGADD45A forms a complex with the scaffold protein AtRACK1A in response to DNA damage.

key messageA novel function of stress sensor mammalian GADD45A homologs, AtGADD45a1 and AtGADD45a2, was identified in Arabidopsis, where they bind to scaffold protein AtRACK1A to regulate the plant DNA damage response. DNA damage, triggered by environmental stressors such as UV radiation and chemical mutagens, threatens plant genomic integrity and necessitates efficient DNA damage response (DDR) pathways. Although mammalian DDR mechanisms are extensively studied, plant DDR remains comparatively underexplored. Here, we characterize Arabidopsis thaliana homologs of mammalian Growth Arrest and DNA Damage 45A (GADD45A), designated AtGADD45a1 and AtGADD45a2. CRISPR/Cas9-generated atgadd45a1 and atgadd45a2 single mutants exhibited hypersensitivity to genotoxins, including Zeocin, mitomycin C, hydroxyurea, and methyl methanesulfonate, characterized by inhibited root elongation and diminished meristematic cell proliferation. Under normal conditions, these mutants displayed slightly delayed flowering and reduced biomass compared to wild-type Col-0, whereas the double mutant was embryo-lethal, indicating functional redundancy. Immunoprecipitation-mass spectrometry revealed the scaffold protein AtRACK1A, serving as a versatile signaling node, as a binding partner. AlphaFold3 predictions highlighted interacting residues in the conserved Ribosomal_L7Ae domain of AtGADD45a1 and WD40 repeats of AtRACK1A. Subsequent investigation demonstrated that AtRACK1A also participates in the genotoxic stress response. RT-qPCR analysis indicated that atgadd45a1-2, atgadd45a2-1, and atrack1a-2 mutants coordinately influenced the expression of DDR genes, such as AtBRCA1, AtRAD51, AtMSH2, and AtKU70. Our findings provide insights into the novel AtGADD45a1/2-AtRACK1A complex in plant-specific DDR.