Cis -regulatory mutations co-opting circadian clock regulation underlie naturally selected extreme trait in Arabidopsis halleri

HEAVY METAL ATPase 4 ( HMA4 ) is required for the naturally selected traits of zinc/cadmium hyperaccumulation and hypertolerance of in Arabidopsis halleri . Cis -regulatory alterations and tandem triplication of AhHMA4 result in substantially elevated transcript levels compared to the closely related non-tolerant non-hyperaccumulator Arabidopsis thaliana . Here we identify cis -regulatory Metal Hyperaccumulation Elements (MHEs) necessary for AhHMA4 promoter 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 all AhHMA4 gene 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 each AhHMA4 gene copy, but not of AtHMA4 , mediates enhanced transcript levels of the reporter and their diel rhythmicity. These functional characteristics are CCA1 -dependent and recapitulated by a synthetic reporter construct placing the MHE2 pair into the AtHMA4 -promoter sequence context, according to the example of the AhHMA4-1 promoter. Consistent with our observations in transgenic reporter lines, AhHMA4 transcript levels follow a diel rhythm in wild-type A. halleri plants. Different from A. halleri , we identify complex repressive functionalities co-localizing with an upstream lncRNA and an intron in the 5’ untranslated region of A. thaliana HMA4 . In summary, our work exemplifies how cis -regulatory mutations contributed to the evolution of extreme physiological traits through the co-option of the circadian clock regulatory network.