The circadian rhythmicity of the universal stress protein is orchestrated by the nuclear clock component CCA1 and 26S-proteasome

Abstract

Arabidopsis Universal Stress Protein (USP; At3g53990) plays critical roles in acting as a stress shield and regulating the expression of the nuclear clock gene, CIRCADIAN CLOCK ASSOCIATED 1(CCA1). To elucidate the reciprocal regulatory interplay between USP and CCA1 proteins, this study employed in silico analysis of USP promoter, uncovering four putative CCA1-binding motifs, and subsequently validating CCA1's interaction with these elements. We investigated the role of CCA1 in regulating USP expression by generating CCA1-deficient mutants (cca1-1) harboring a PUSP:LUC reporter. These mutants exhibited a pronounced enhancement in USP amplitude, implicating CCA1 as a negative regulator of USP expression. Subsequently, we investigated the protein dynamics of USP, utilizing plants expressing HA-tagged-USP prepared in usp background (PUSP:USP-HA/ usp). Immunoblotting unveiled rhythmic oscillations in USP abundance, prompting us to delineate the regulatory mechanisms governing USP's circadian rhythm. Treatment of plants with protein synthesis and proteasome inhibitors revealed that USP abundance decreases during the night through proteasome-mediated degradation. Leveraging the STRING protein-interaction databases, we identified the culprit behind USP degradation and validated PUB35 as the USP-specific E3-ligase. Our findings elucidate the intricate reciprocal interplay between the stress-responsive protein and the circadian clock machinery, illuminating the mechanistic underpinnings that govern the daily oscillations in USP abundance.