Cis-regulatory elements co-opting core circadian clock regulator CCA1 underlie enhanced expression of HMA4 for metal hyperaccumulation in Arabidopsis halleri.

Authors

Leonardo Castanedo, Justyna Cebula, Cécile Nouet, Julien Spielmann, Nedežda Janina, Marc Hanikenne, Ute Krämer

Published in

Plant communications. Pages 101540. Oct 03, 2025. Epub Oct 03, 2025.

Abstract

The naturally selected extreme traits of zinc/cadmium hyperaccumulation and hypertolerance in Arabidopsis halleri depend on strongly elevated HEAVY METAL ATPase 4 (HMA4) transcript levels compared to the closely related Arabidopsis thaliana. This is governed in cis, meaning that upstream AhHMA4 sequences are sufficient, as previously demonstrated using reporter gene fusions stably introduced into both A. halleri and A. thaliana. However, the underlying cis-regulatory mutations specific to A. halleri have remained unknown. Here we identify cis-regulatory Metal Hyperaccumulation Elements (MHE) that contribute to the increased activity of the promoters of the three tandem AhHMA4 gene copies by examining lines stably transformed with deletion and mutant variants of reporter constructs. MHE1 (consensus TGTAAC) functions in distal regions of AhHMA4 promoters, and all three AhHMA4 gene copies share a proximal upstream pair of MHE2 (consensus AAATATCT, Evening Element, EE). The EE is a known target of Arabidopsis CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), a transcription factor that mediates light-regulated gene expression and operates in the circadian clock. We show that the elevated activity of the AhHMA4-1 promoter depends on MHE2 in cis and CCA1 in trans, and it is recapitulated by site-directed mutagenesis generating an intact pair of MHE2 in the A. thaliana HMA4 promoter sequence. HMA4 transcript levels show diel rhythmicity in A. halleri but not A. thaliana. In summary, we identify the causal cis-regulatory elements which underlie enhanced HMA4 transcript levels critical for a naturally selected extreme trait syndrome and function by co-opting a regulator of diel and seasonal transcriptional rhythms.

PMID:
41046334
Bibliographic data and abstract were imported from PubMed on 05 Oct 2025.

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