As the global temperature gradually increases, thermotolerance is vital to the growth and survival for plants. Ubiquitin-mediated protein degradation is a central regulator of many key cellular and physiological processes, including responses to biotic and abiotic stresses. E3 Ubiquitin-ligases, as the major components in the ubiquitination pathway, confer specificity of substrate recognition. Herein, we report that AtPUB48 expression was induced by heat stress, including basal and acquired thermotolerance. AtPUB48-overexpressing lines (OEs) of plants were generated to detect the functions of AtPUB48 in the heat response signaling pathway in Arabidopsis. Seeds of Atpub48-2 mutant had a lower germination rate than those of wild-type (WT) and OE plants when suffered from high temperatures. On the contrary, overexpression of AtPUB48 in Arabidopsis enhanced basal and acquired thermotolerance in seed germination and seedling growth. Moreover, the transcript expression levels of several heat-related downstream genes were highly improved in the OE lines under heat stress, although there were lower levels in the Atpub48-2 mutant compared with that of WT. An in vitro ubiquitination assay confirmed that AtPUB48 with U-box and ARM-repeats functioned as an E3 ubiquitin ligase. The subcellular localization showed that AtPUB48 localized to the nucleus. Collectively, these data imply that AtPUB48 acts as a novel regulator in the heat response signaling pathway. AtPUB48 may target the unknown substrate receptor to 26S proteasome proteolysis. (C) 2018 Elsevier Inc. All rights reserved.