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bioRxiv · 10.1101/2024.10.14.618290

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

Castanedo, L.·Cebula, J.·Nouet, C.·Spielmann, J.·Hanikenne, M.·Kraemer, U.
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Abstract

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.

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Castanedo, L., Cebula, J., Nouet, C., Spielmann, J., Hanikenne, M., Kraemer, U.. 2024-10-17. Cis-regulatory mutations co-opting circadian clock regulation underlie naturally selected extreme trait in Arabidopsis halleri. https://doi.org/10.1101/2024.10.14.618290

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