Circadian clock-regulated expression of an RNA-binding protein in Arabidopsis: characterisation of a minimal promoter element

Abstract

The Atgrp7 transcript encodes a clock-regulated, glycine-rich, RNA-binding protein in Arabidopsis thaliana and shows a circadian variation in steady-state abundance. Constitutive overexpression of its product, AtGRP7, in transgenic Arabidopsis plants depresses the oscillations of the endogenous Atgrp7 transcript, indicating that both the transcript and the protein are part of a clock-regulated negative feedback circuit. Here we characterise the upstream region of the Atgrp7 gene in order to begin to dissect the molecular basis of this oscillating autoregulatory feedback loop. Fusion of a 1.5-kb promoter fragment to the beta-glucuronidase (gus) reporter gene leads to circadian oscillations in the level of the gus transcript in transgenic Arabidopsis plants, with highest levels in the evening, indicating that transcription of the Atgrp7 gene is rhythmically activated by the endogenous circadian clock. A 265-bp fragment upstream of the transcription start site is necessary for high-amplitude Atgrp7 cycling. Within this region, a 56-bp clock-responsive element that confers a low-amplitude circadian oscillation (approximately threefold) with peak abundance in the early evening maps between positions -112 and -57. Another element necessary for augmenting the amplitude of the oscillation lies between -178 and -264. Genetic crosses between a line bearing a promoter-gus fusion and plants that overexpress AtGRP7 show that the promoter by itself does not mediate the negative feedback of AtGRP7 on the oscillations of its own transcript.