[en] AbstractHEAVY METAL ATPase 4(HMA4) is required for the naturally selected traits of zinc/cadmium hyperaccumulation and hypertolerance of inArabidopsis halleri.Cis-regulatory alterations and tandem triplication ofAhHMA4result in substantially elevated transcript levels compared to the closely related non-tolerant non-hyperaccumulatorArabidopsis thaliana. Here we identifycis-regulatory Metal Hyperaccumulation Elements (MHEs) necessary forAhHMA4promoter activity, employing sequence comparisons and motif elicitation analyses combined with progressive deletions and site-directed mutagenesis of promoter-reporter constructs. We report that the promoters of allAhHMA4gene copies share a distal MHE1 (consensus TGTAAC), and a proximal pair of MHE2s identical or highly similar to the Evening Element (AAAATATCT). Evening elements are known binding sites of Arabidopsis CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), a phytochrome-regulated transcription factor in the core circadian clock. We show that the promoter of eachAhHMA4gene copy, but not ofAtHMA4, mediates enhanced transcript levels of the reporter and their diel rhythmicity. These functional characteristics areCCA1-dependent and recapitulated by a synthetic reporter construct placing the MHE2 pair into theAtHMA4-promoter sequence context, according to the example of theAhHMA4-1promoter. Consistent with our observations in transgenic reporter lines,AhHMA4transcript levels follow a diel rhythm in wild-typeA. halleriplants. Different fromA. halleri, we identify complex repressive functionalities co-localizing with an upstream lncRNA and an intron in the 5’ untranslated region ofA. thaliana HMA4. In summary, our work exemplifies howcis-regulatory mutations contributed to the evolution of extreme physiological traits through the co-option of the circadian clock regulatory network.
